Composition for reducing frequency of urination, method of making and use thereof

ABSTRACT

Pharmaceutical compositions for reducing frequency of urination are disclosed. The pharmaceutical compositions comprise one or more prostaglandin pathway inhibitors and a pharmaceutically acceptable carrier. Also disclosed are methods of making and using the pharmaceutical compositions.

This application is a divisional application of U.S. application Ser.No. 14/975,332, filed Dec. 18, 2015 which is a continuation-in-partapplication of U.S. application Ser. No. 14/298,511, filed on Jun. 6,2014, now U.S. Pat. No. 9,532,959 . The entirety of the aforementionedapplication is incorporated herein by reference.

FIELD

The present application generally relates to methods and compositionsfor inhibiting the smooth muscles of the urinary bladder and, inparticular, to methods and compositions for reducing the frequency ofurination.

BACKGROUND

The detrusor muscle is a layer of the urinary bladder wall made ofsmooth muscle fibers arranged in spiral, longitudinal, and circularbundles. When the bladder is stretched, this signals the parasympatheticnervous system to contract the detrusor muscle. This encourages thebladder to expel urine through the urethra.

For the urine to exit the bladder, both the autonomically controlledinternal sphincter and the voluntarily controlled external sphinctermust be opened. Problems with these muscles can lead to incontinence. Ifthe amount of urine reaches 100% of the urinary bladder's absolutecapacity, the voluntary sphincter becomes involuntary and the urine willbe ejected instantly.

The human adult urinary bladder usually holds about 300-350 ml of urine(the working volume), but a full adult bladder may hold up to about 1000ml (the absolute volume), varying among individuals. As urineaccumulates, the ridges produced by folding of the wall of the bladder(rugae) flatten and the wall of the bladder thins as it stretches,allowing the bladder to store larger amounts of urine without asignificant rise in internal pressure.

In most individuals, the desire to urinate usually starts when thevolume of urine in the bladder reaches around 200 ml. At this stage itis easy for the subject, if desired, to resist the urge to urinate. Asthe bladder continues to fill, the desire to urinate becomes strongerand harder to ignore. Eventually, the bladder will fill to the pointwhere the urge to urinate becomes overwhelming, and the subject will nolonger be able to ignore it.

In some individuals, this desire to urinate starts when the bladder isless than 100% full in relation to its working volume. Such increaseddesire to urinate may interfere with normal activities, including theability to sleep for sufficient uninterrupted periods of rest. In somecases, this increased desire to urinate may be associated with medicalconditions such as benign prostate hyperplasia or prostate cancer inmen, or pregnancy in women. However, increased desire to urinate alsooccurs in individuals, both male and female, who are not affected byanother medical condition.

In some individuals, such as in children, involuntary urination (e.g.,bed wetting) may occur as a result of lack of control to the bladdermuscle. In other individuals, involuntary urination (e.g., urinaryincontinence) may occur as a result from an underlying medicalcondition.

Accordingly, there exists a need for compositions and methods for thetreatment of male and female subjects who suffer from an undesiredfrequency of urination.

SUMMARY

One aspect of the present application relates to a method formanufacturing a pharmaceutical composition for reducing the frequency ofurination. In some embodiments, the method comprises the steps offorming a first mixture comprising a first active ingredient formulatedfor immediate release and a second active ingredient formulated forextended release; coating the first mixture with a delayed releasecoating to form a core structure; coating the core structure with asecond mixture comprising a third active ingredient formulated forimmediate release and a fourth active ingredient formulated for extendedrelease, wherein at least one of the first, second, third and fourthactive ingredients comprises a prostaglandin pathway inhibitor. In someembodiments, the prostaglandin pathway inhibitor is a prostaglandin (PG)inhibitor, or a prostaglandin transporter (PGT) inhibitor, or aprostaglandin receptor (PGR) inhibitor.

In other embodiments, the method comprises the steps of forming a corestructure comprising a first active ingredient formulated for immediaterelease and a second active ingredient formulated for extended release;coating the core structure with a delayed release coating to form acoated core structure; mixing the coated core structure with a thirdactive ingredient formulated for immediate release and a fourth activeingredient formulated for extended release to form a final mixture; andpreparing a dosage form with the final mixture, wherein at least one ofthe first, second, third and fourth active ingredients comprises aprostaglandin pathway inhibitor.

In other embodiments, the method comprises the steps of forming a corestructure comprising a first active ingredient formulated for immediaterelease and a second active ingredient formulated for extended release;coating the core structure with a delayed release coating to form acoated core structure; coating the coated core structure with a thirdactive ingredient formulated for extended release to form anextended-release layer coated core structure; and coating theextended-release layer coated core structure with a fourth activeingredient, wherein at least one of the first, second, third and fourthactive ingredients comprises a prostaglandin pathway inhibitor.

Another aspect of the present application relates to a pharmaceuticalcomposition for treating a condition that results in undesired frequencyof urination. In some embodiments, the pharmaceutical compositioncomprises: a first component comprising an immediate-releasesubcomponent and an extended-release subcomponent, wherein the firstcomponent is formulated to release the subcomponents immediately afteradministration; and a second component comprising an immediate-releasesubcomponent and an extended-release subcomponent, wherein the secondcomponent is formulated for a delayed-release of the subcomponents,wherein at least one of subcomponents in the first component and thesecond component comprises an active ingredient comprising aprostaglandin pathway inhibitor.

In other embodiments, the pharmaceutical composition comprises: a firstcomponent comprising an immediate-release subcomponent, wherein theimmediate-release subcomponent comprises an active ingredient comprisingone or more agents selected from the group consisting of analgesicagents and prostaglandin pathway inhibitors, wherein the first componentis formulated to release its subcomponent immediately after oraladministration; and a second component comprising an immediate-releasesubcomponent and an extended-release subcomponent, wherein the secondcomponent is formulated to release its subcomponent after gastricemptying of the second component, wherein at least one of thesubcomponents in the first and the second components comprises an activeingredient comprising one or more agents selected from the groupconsisting of analgesic agents and prostaglandin pathway inhibitors.

In other embodiments, the pharmaceutical composition comprises: animmediate-release component comprising acetaminophen and an NSAID, eachin an amount of 5-2000 mg; and an extended-release component comprisingacetaminophen and an NSAID, each in an amount of 5-2000 mg, wherein theimmediate-release component, or the extended-release component, or both,further comprise a prostaglandin pathway inhibitor.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are diagrams showing that analgesics regulate expressionof co-stimulatory molecules by Raw 264 macrophage cells in the absence(FIG. 1A) or presence (FIG. 1B) of LPS. Cells were cultured for 24 hrsin the presence of analgesic alone or together with Salmonellatyphimurium LPS (0.05 μg/ml). Results are mean relative % of CD40+CD80+cells.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. The present invention is notintended to be limited to the embodiments shown, but is to be accordedthe broadest possible scope consistent with the principles and featuresdisclosed herein.

As used herein, the term “prostaglandin (PG)” refers to a group of lipidcompounds that are derived enzymatically from fatty acids and have avariety of physiological effects, such as regulating the contraction andrelaxation of smooth muscle tissue, in the animal body. Everyprostaglandin contains 20 carbon atoms, including a 5-carbon ring.Examples of prostaglandin include, prostaglandin E₁ (PGE₁),prostaglandin E₂ (PGE₂), Prostaglandin D₂, prostaglandin I₂ (PGI₂,prostacyclin), and prostaglandin F_(2α) (PGF_(2α)).

As used herein, the term “prostaglandin (PG) pathway inhibitor” refersto agents that interact directly or indirectly with one or morecomponents involved in the synthesis or action of PG on a target tissue,and interfere with either the levels or the ultimate actions ofprostaglandin on the target tissue. PG pathway inhibitors include, butare not limited to, PG inhibitors, prostaglandin transporter (PGT)inhibitors and prostaglandin receptor (PGR) inhibitors. The term “PGpathway inhibitor,” however, does not include the analgesics definedbelow.

The term “PG inhibitors,” as used herein, include but are not limitedto, inhibitors of PG synthesis and inhibitors of PG activity. The term“inhibitors of PG synthesis,” as used herein, refers to agents thatinhibit the production of prostaglandin, such as agents that inhibit theexpression or activity of phospholipase A2, the prostaglandin synthasesand the tissue specific isomerases and synthase enzymes such as:Thromboxane synthase, PGF synthase, cytosolic PG synthase (cPGES),prostaglandin I synthase (PGIS) and the microsomal PGES enzymes (mPGES).Examples of PG synthesis inhibitors include flunixin meglumine. As usedherein, the terms “inhibitors of PG synthesis” and “PG synthesisinhibitors” do not include the analgesics defined below.

The term “inhibitors of PG activity,” as used herein, refers to agentsthat antagonize the action of prostaglandin itself by any means. Agentsthat interfere solely with the synthesis of prostaglandins, such as byinterfering with the action of prostaglandin synthases, but which do notinterfere with the action of prostaglandins are not included within thedefinition of inhibitors of PG activity as used in this specification.

The term “PGT inhibitors,” as used herein, refers to agents thatinhibits the expression or the activity of PG transporters, such as ATPdependent multi-drug resistance (MDR) transporter-4, or other MDRchannels such as ABCC1, ABCC2, ABCC3, ABCC6, ABCG2 and ABCB11. Examplesof PGT inhibitors that inhibit PGT activity include, but are not limitedto, compounds that inhibit MDR membrane pumps, such as triazinecompounds, verapamil, and calcium channel blockers; channels includequinidines, ketoconazole, itraconazole, azithromycin, valspodar,cyclosporine, elacridar, fumitremorgin-C, gefitinib and erythromycin.Examples of PGT inhibitors that inhibit PGT expression include, but arenot limited to, agents which control the transcription of the MDR genesby targeting the promoter region and/or transcription factors which bindto the promoter or other gene control regions. The term “PGRinhibitors,” as used herein, refers to agents that inhibits the activityor expression of PGRs. In some embodiments, the PGRs comprise Eprostanoid receptor EP1, EP2, EP3, and EP4 subtypes of the PGE receptor;PGD receptor (DP1); PGF receptor (FP); PGI receptor (IP); andthromboxane receptor (TP). Two additional isoforms of the human TP (TPαand TPβ) and FP (FPA and FPB) and eight EP3 variants are generatedthrough alternative splicing, which differ only in their C-terminaltails. In some embodiments, the PGRs further comprise a Gprotein-coupled receptor termed chemo-attractant receptor-homologousmolecule (CRHME). In other embodiments, the PGRs include all of thereceptors that activate rhodopsin-like 7-transmembrane-spanning Gprotein-coupled receptors.

Examples of PGR activity inhibitors include, but are not limited to,anti-PGR antibodies and any agent that inhibits the G-protein coupledreceptor signaling pathway. PGR expression inhibitors include agentsthat inhibit PGR expression at the transcriptional level, translationallevel or post transcriptional level. Examples of PGR expressioninhibitors include, but are not limited to, anti-PGR siRNA and mi RNAs.

As used herein, the term “an effective amount” means an amount necessaryto achieve a selected result.

As used herein, the term “analgesic” refers to agents, compounds ordrugs used to relieve pain and inclusive of anti-inflammatory compounds.Exemplary analgesic and/or anti-inflammatory agents, compounds or drugsinclude, but are not limited to, non-steroidal anti-inflammatory drugs(NSAIDs), salicylates, aspirin, salicylic acid, methyl salicylate,diflunisal, salsalate, olsalazine, sulfasalazine, para-aminophenolderivatives, acetanilide, acetaminophen, phenacetin, fenamates,mefenamic acid, meclofenamate, sodium meclofenamate, heteroaryl aceticacid derivatives, tolmetin, ketorolac, diclofenac, propionic acidderivatives, ibuprofen, naproxen sodium, naproxen, fenoprofen,ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicam derivatives,piroxicam, meloxicam, tenoxicam, ampiroxicam, droxicam, pivoxicam,pyrazolon derivatives, phenylbutazone, oxyphenbutazone, antipyrine,aminopyrine, dipyrone, coxibs, celecoxib, rofecoxib, nabumetone,apazone, indomethacin, sulindac, etodolac, isobutylphenyl propionicacid, lumiracoxib, etoricoxib, parecoxib, valdecoxib, tiracoxib,etodolac, darbufelone, dexketoprofen, aceclofenac, licofelone,bromfenac, loxoprofen, pranoprofen, piroxicam, nimesulide, cizolirine,3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one,meloxicam, lornoxicam, d-indobufen, mofezolac, amtolmetin, pranoprofen,tolfenamic acid, flurbiprofen, suprofen, oxaprozin, zaltoprofen,alminoprofen, tiaprofenic acid, pharmacological salts thereof, hydratesthereof, and solvates thereof

As used herein, the term “coxib” refers to a compound or composition ofcompounds that is capable of inhibiting the activity or expression ofCOX1 and COX2 enzymes.

As used herein, the term “derivative” refers to a chemically modifiedcompound wherein the modification is considered routine by the ordinaryskilled chemist, such as an ester or an amide of an acid, or protectinggroups such as a benzyl group for an alcohol or thiol, or atert-butoxycarbonyl group for an amine.

As used herein, the term “analogue” refers to a compound which comprisesa chemically modified form of a specific compound or class thereof andwhich maintains the pharmaceutical and/or pharmacological activitiescharacteristic of said compound or class.

As used herein, the term “pharmaceutically acceptable salts” refer toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, sulfamic acid, phosphoric acid, nitric acid, and the like and thesalts prepared from organic acids such as acetic acid, propionic acid,succinic acid, glycolic acid, stearic acid, lactic acid, malic acid,tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid,hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid,salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid,toluensulfonic acid, methanesulfonic acid, ethane dislfonic acid, oxalicacid, isethionic acid, and the like.

As used herein, the phrase “pharmaceutically acceptable” is used withreference to compounds, materials, compositions and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problems orcomplications commensurate with a reasonable benefit/risk ratio.

The term “immediate-release” is used herein with reference to a drugformulation that does not contain a dissolution rate controllingmaterial. There is substantially no delay in the release of the activeagents following administration of an immediate-release formulation. Animmediate-release coating may include suitable materials immediatelydissolving following administration so as to release the drug contentstherein. In some embodiments, the term “immediate-release” is used withreference to a drug formulation that releases the active ingredient inless than 10 min, 20 min, 30 min, 40 min 50 min, 60 min, 90 min or 120min after administration into a patient.

As used herein, the term “extended-release,” also known assustained-release (SR), sustained-action (SA), time-release (TR),controlled-release (CR), modified release (MR), or continuous-release(CR), refers to a mechanism used in medicine tablets or capsules todissolve slowly and release the active ingredient over time. Theadvantages of extended-release tablets or capsules are that they canoften be taken less frequently than immediate-release formulations ofthe same drug and that they keep steadier levels of the drug in thebloodstream, thus extending the duration of the drug action and loweringthe peak amount of drug in the bloodstream. In some embodiments, theterm “extended-release” refers to a release profile that the activeingredient in a tablet or capsule is released over a period of 2, 3, 4,5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or 24 hour, eithercontinuously or in pulses after administration into a patient.

As used herein, the term “delayed-release” refers to a drug releaseprofile that the release of the active ingredient(s) of a pharmaceuticalcomposition is delayed or postponed for a given period of time (e.g., 1,2, 3, 4 or 5 hours, or after stomach) after administration of thepharmaceutical composition.

As used herein, the term “delayed-extended-release” refers to a drugrelease profile that the release of the active ingredient(s) of apharmaceutical composition is delayed or postponed for a given period oftime (e.g., the lag period of 1, 2, 3, 4 or 5 hours, or after stomach)after administration of the pharmaceutical composition. Once the releasestarts, the active ingredient(s) is released slowly over time (e.g.,over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or24 hour), either continuously or in pulses.

Method for Reducing Frequency of Urination

One aspect of the present application relates to a method for reducingfrequency of urination by administering to a subject having a conditionthat results in an undesired frequency of urination an effective amountof a pharmaceutical composition. The pharmaceutical compositioncomprises one or more PG pathway inhibitors and a pharmaceuticallyacceptable carrier. Conditions that result in an undesired frequency ofurination include, but are not limited to, nocturia, overactive bladder,urinary incontinence and bed wetting.

In some embodiments, the PG inhibitor is an inhibitor of PG synthetase.Examples of inhibitors of PG synthesis include, but are not limited to,inhibitors of PG synthetase.(this is redundant) In other embodiments,the PG inhibitor is an inhibitor of PG activity. Examples of inhibitorsof PG activity include, but are not limited to agents which block thebinding of PG to any of its receptors: EP1, EP2, EP3, EP4. DP1, DP2,FP2, IP and TP. Examples of these types of inhibitors include, but arenot limited to, the IP receptor inhibitor developed by Roche: RO3244019,ONO-85-39 which is an EP1 receptor antagonist, the dual EP1 and EP2receptor antagonist AH 6809, and the EP4 antagonist. RQ-15986. Incertain embodiments, the one or more PG pathway inhibitors comprise aPGT inhibitor. In some embodiments, the PGT inhibitor is a PGT activityinhibitor. Examples of PGT activity inhibitor include, but are notlimited to, anti-PGT antibodies, and any known compound which caninhibit the ATP-dependent Multi drug resistance transporter-4 or relatedMDR pumps that are shown to transport PGs. In other embodiments, the PGTinhibitor is a PGT expression inhibitor. Examples of PGT expressioninhibitor include, but are not limited to, anti-PGT siRNA, antisenseRNAs that target PGT mRNA, and agents which control the transcription ofthe gene by influencing DNA methylation and or chromatin modification.

In some embodiments, the one or more PG pathway inhibitors comprise aninhibitor that targets both the COX active site and the PDX active sitewhich are contained in both COX1 and COX2. In other embodiments, the oneor more PG pathway inhibitors comprise an inhibitor that inhibits thePGE₂ pathway.

In certain embodiments, the one or more PG pathway inhibitors comprise aPGR inhibitor. PGRs are G-protein-coupled receptors containing seventransmembrane domains. Examples of PGR include, EP1, EP2, EP3, EP4, DP1,DP2, FP, IP1, IP2 , CRTH2 and TP receptors. In some embodiments, the oneor more PG pathway inhibitors comprise an inhibitor that inhibits any ofthe PG receptors listed above. In some embodiments, the PGR inhibitor isa PGR activity inhibitor. Examples of PGR activity inhibitor include,but are not limited to, anti-PGR antibodies. In some embodiments, thePGR inhibitor is an inhibitor of PGE2 receptor activity, such as EP1activity inhibitor, EP2 activity inhibitor, EP3 activity inhibitor orEP4 activity inhibitor.

In other embodiments, the PGR inhibitor is a PGR expression inhibitor.Examples of PGR expression inhibitor include, but are not limited to,anti-PGR siRNA, antisense RNAs that target PGR mRNA, or agents whichcontrol the transcription of the gene by influencing DNA methylation andor chromatin modification. In some embodiments, the PGR expressioninhibitor is an inhibitor of PGE2 receptor expression, such as EP1expression inhibitor, EP2 expression inhibitor, EP3 expression inhibitoror EP4 expression inhibitor. In some embodiments, the one or more PGpathway inhibitors comprise a small molecule inhibitor. As used herein,the term “small molecule inhibitor” refers to inhibitors having amolecular weight of 1000 dalton or less.

In some embodiments, the PG pathway inhibitor comprises a shortinterfering RNA (siRNA). An siRNA is a double-stranded RNA that can beengineered to induce sequence-specific post-transcriptional genesilencing of mRNAs corresponding to a component of the PG pathway.siRNAs exploit the mechanism of RNA interference (RNAi) for the purposeof “silencing” gene expression of e.g., targeted PGE₂ receptor genes.This “silencing” was originally observed in the context of transfectingdouble stranded RNA (dsRNA) into cells. Upon entry therein, the dsRNAwas found to be cleaved by an RNase III-like enzyme, Dicer, into doublestranded small interfering RNAs (siRNAs) 21-23 nucleotides in lengthcontaining 2 nucleotide overhangs on their 3′ ends. In an ATP dependentstep, the siRNAs become integrated into a multi-subunit RNAi inducedsilencing complex (RISC) which presents a signal for AGO2-mediatedcleavage of the complementary mRNA sequence, which then leads to itssubsequent degradation by cellular exonucleases.

In some embodiments, the PG pathway inhibitor comprises a syntheticsiRNA or other class of small RNA targeting a PG synthase RNA, a PGT RNAor a PGR RNA in the target cell/tissue. Synthetically produced siRNAsstructurally mimic the types of siRNAs normally processed in cells bythe enzyme Dicer. Synthetically produced siRNAs may incorporate anychemical modifications to the RNA structure that are known to enhancesiRNA stability and functionality. For example, in some cases, thesiRNAs may be synthesized as a locked nucleic acid (LNA)-modified siRNA.An LNA is a nucleotide analogue that contains a methylene bridgeconnecting the 2′-oxygen of the ribose with the 4′ carbon. The bicyclicstructure locks the furanose ring of the LNA molecule in a 3′-endoconformation, thereby structurally mimicking the standard RNA monomers.

In other embodiments, the PG pathway inhibitor comprises an expressionvector engineered to transcribe a short double-stranded hairpin-like RNA(shRNA) that is processed into a targeted siRNA inside the cell. TheshRNAs can be cloned in suitable expression vectors using kits, such asAmbion's SILENCER® siRNA Construction Kit, Imgenex's GENESUPPRESSOR™Construction Kits and Invitrogen's BLOCK-IT™ inducible RNAi plasmid andlentivirus vectors. Synthetic siRNAs and shRNAs may be designed usingwell known algorithms and synthesized using a conventional DNA/RNAsynthesizer.

In some embodiments, the secondary active agent comprises an antisenseoligonucleotide or polynucleotide capable of inhibiting the expressionof a component of the PG pathway. The antisense oligonucleotide orpolynucleotide may comprise a DNA backbone, RNA backbone or chemicalderivative thereof In one embodiment, the antisense oligonucleotide orpolynucleotide comprises a single stranded antisense oligonucleotide orpolynucleotide targeting for degradation. In certain embodiments, theanti-inflammatory agent comprises a single stranded antisenseoligonucleotide complementary to the mRNA sequence of a component of thePG pathway. The single stranded antisense oligonucleotide orpolynucleotide may be synthetically produced or it may be expressed froma suitable expression vector. The antisense nucleic acid is designed tobind via complementary binding to the mRNA sense strand so as to promoteRNase H activity, which leads to degradation of the mRNA. Preferably,the antisense oligonucleotide is chemically or structurally modified topromote nuclease stability and/or increased binding.

In some embodiments, the antisense oligonucleotides are modified toproduce oligonucleotides with nonconventional chemical or backboneadditions or substitutions, including, but not limited to, peptidenucleic acids (PNAs), locked nucleic acids (LNAs), morpholino backbonednucleic acids, methylphosphonates, duplex stabilizing stilbene orpyrenyl caps, phosphorothioates, phosphoroamidates, phosphotriesters andthe like. By way of example, the modified oligonucleotides mayincorporate or substitute one or more of the naturally occurringnucleotides with an analog; internucleotide modifications incorporating,for example, uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.) or charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.); modificationsincorporating intercalators (e.g., acridine, psoralen, etc.), chelators(e.g., metals, radioactive metals, boron, oxidative metals, etc.) oralkylators and/or modified linkages (e.g., alpha anomeric nucleic acids,etc.).

In some embodiments, the single stranded oligonucleotides are internallymodified to include at least one neutral charge in its backbone. Forexample, the oligonucleotide may include a methylphosphonate backbone orpeptide nucleic acid (PNA) complementary to the target-specificsequence. These modifications have been found to prevent or reducehelicase-mediated unwinding. The use of uncharged probes may furtherincrease the rate of hybridization to polynucleotide targets in a sampleby alleviating the repulsion of negatively-charges nucleic acid strandsin classical hybridization.

PNA oligonucleotides are uncharged nucleic acid analogs for which thephosphodiester backbone has been replaced by a polyamide, which makesPNAs a polymer of 2-aminoethyl-glycine units bound together by an amidelinkage. PNAs are synthesized using the same Boc or Fmoc chemistry asare use in standard peptide synthesis. Bases (adenine, guanine, cytosineand thymine) are linked to the backbone by a methylene carboxyl linkage.Thus, PNAs are acyclic, achiral and neutral. Other properties of PNAsare increased specificity and melting temperature as compared to nucleicacids, capacity to form triple helices, stability at acid pH,non-recognition by cellular enzymes like nucleases, polymerases, etc.

Methylphosphonate-containing oligonucleotides are neutral DNA analogscontaining a methyl group in place of one of the non-bonding phosphoryloxygens. Oligonucleotides with methylphosphonate linkages were among thefirst reported to inhibit protein synthesis via anti-sense blockade oftranslation.

In some embodiments, the phosphate backbone in the oligonucleotides maycontain phosphorothioate linkages or phosphoroamidates. Combinations ofsuch oligonucleotide linkages are also within the scope of the presentinvention.

In other embodiments, the oligonucleotide may contain a backbone ofmodified sugars joined by phosphodiester internucleotide linkages. Themodified sugars may include furanose analogs, including but not limitedto 2-deoxyribofuranosides, α-D-arabinofuranosides,α-2′-deoxyribofuranosides and 2′,3′-dideoxy-3′-aminoribofuranosides. Inalternative embodiments, the 2-deoxy-β-D-ribofuranose groups may bereplaced with other sugars, for example, β-D-ribofuranose. In addition,β-D-ribofuranose may be present wherein the 2-OH of the ribose moiety isalkylated with a C1-6 alkyl group (2-(O—C1-6 alkyl) ribose) or with aC2-6 alkenyl group (2-(O—C2-6 alkenyl) ribose) or is replaced by afluoro group (2-fluororibose).

Related oligomer-forming sugars include those used in locked nucleicacids (LNA) as described above. Exemplary LNA oligonucleotides includemodified bicyclic monomeric units with a 2′-O-4′-C methylene bridge,such as those described in U.S. Pat. No. 6,268,490.

Chemically modified oligonucleotides may also include, singly or in anycombination, 2′-position sugar modifications, 5-position pyrimidinemodifications (e.g., 5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-napthylcarboxyamide)-2′-deoxyuridine,5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine), 8-positionpurine modifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo- or 5-iodo-uracil, methylations,unusual base-pairing combinations, such as the isobases isocytidine andisoguanidine and the like.

In some embodiments, the one or more PG pathway inhibitors comprise aribozyme capable of inhibiting the expression of a component of the PGpathway. Ribozymes are nucleic acid molecules that are capable ofcatalyzing a chemical reaction, either intramolecularly orintermolecularly. Ribozymes are thus catalytic nucleic acid. It ispreferred that the ribozymes catalyze intermolecular reactions. Thereare a number of different types of ribozymes that catalyze nuclease ornucleic acid polymerase type reactions which are based on ribozymesfound in natural systems, such as hammerhead ribozymes, hairpinribozymes and tetrahymena ribozymes. There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo. Preferred ribozymescleave RNA or DNA substrates and more preferably cleave RNA substrates,such as mRNAs of components of the PG pathway. Ribozymes typicallycleave nucleic acid substrates through recognition and binding of thetarget substrate with subsequent cleavage. This recognition is oftenbased mostly on canonical or non-canonical base pair interactions. Thisproperty makes ribozymes particularly good candidates for targetspecific cleavage of nucleic acids because recognition of the targetsubstrate is based on the target substrates sequence.

In some embodiments, the one or more PG pathway inhibitors comprisetriplex forming oligonucleotide capable of inhibiting the expression ofa component of the PG pathway. Triplex forming oligonucleotides (TFOs)are molecules that can interact with either double-stranded and/orsingle-stranded nucleic acids, including both coding and non-codingregions in genomic DNA targets. When TFOs interact with a target region,a structure called a triplex is formed, in which there are three strandsof DNA forming a complex dependant on both Watson-Crick and Hoogsteenbase-pairing. TFOs can bind target regions with high affinity andspecificity. In preferred embodiments, the triplex forming moleculesbind the target molecule with a Kd less than 10-6, 10-8, 10-10 or 10-12.Exemplary TFOs for use in the present invention include PNAs, LNAs andLNA modified PNAs, such as Zorro-LNAs.

In some embodiments, the one or more PG pathway inhibitors comprise anexternal guide sequences (EGS). External guide sequences (EGSs) aremolecules that bind a target nucleic acid molecule forming a complex.This complex is recognized by RNase P, which cleaves the targetmolecule. EGSs can be designed to specifically target an mRNA moleculeof choice. RNAse P aids in processing transfer RNA (tRNA) within a cell.Bacterial RNAse P can be recruited to cleave virtually any RNA sequenceby using an EGS that causes the target RNA:EGS complex to mimic thenatural tRNA substrate. Similarly, eukaryotic EGS/RNAse P-directedcleavage of RNA can be utilized to cleave desired targets withineukaryotic cells.

In other embodiments, the one or more PG pathway inhibitors comprise abiomolecule. As used herein, the term “biomolecule” is any molecule thatis produced by a living organism, including large macromolecules such asproteins, polysaccharides, lipids, and nucleic acids, as well as smallmolecules such as primary metabolites, secondary metabolites, andnatural products.

In other embodiments, the one or more PG pathway inhibitors comprisetarget neutralization agents. As used herein, the term “targetneutralization agent” refers to antibodies, fragments of antibodies, orany other non-antibody peptide or synthetic binding molecule, such as anaptamer or synbody, which is capable of specifically binding directly orindirectly to a component of the PG pathway so as to interfere with theultimate actions of prostaglandin on the target tissue.

The target neutralization agents may be produced by any conventionalmethod for generating high-affinity binding ligands, including SELEX,phage display and other methodologies, including combinatorial chemistryand/or high throughput methods known to those of skill in the art.

An aptamer is a nucleic acid version of an antibody that comprises aclass of oligonucleotides that can form specific three dimensionalstructures exhibiting high affinity binding to a wide variety of cellsurface molecules, proteins and/or macromolecular structures. Aptamersare commonly identified by an in vitro method of selection sometimesreferred to as Systematic Evolution of Ligands by EXponential enrichmentor “SELEX.” SELEX typically begins with a very large pool of randomizedpolynucleotides which is generally narrowed to one aptamer ligand permolecular target. Typically, aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets.

An aptamer can be chemically linked or conjugated to the above describednucleic acid inhibitors to form targeted nucleic acid inhibitors, suchas aptamer-siRNA chimeras. An aptamer-siRNA chimera contains a targetingmoiety in the form of an aptamer which is linked to an siRNA. When usingan aptamer-siRNA chimera, it is preferable to use a cell internalizingaptamer. Upon binding to specific cell surface molecules, the aptamercan facilitate internalization into the cell where the nucleic acidinhibitor acts. In one embodiment both the aptamer and the siRNAcomprises RNA. The aptamer and the siRNA may comprise any nucleotidemodifications as further described herein. Preferably, the aptamercomprises a targeting moiety specifically directed to binding cellsexpressing the chemokine-, cytokine- and/or receptor target genes, suchas lymphoid, epithelial cell and/or endothelial cells.

Synbodies are synthetic antibodies produced from libraries comprised ofstrings of random peptides screened for binding to target proteins ofinterest.

Target neutralization agents, including aptamers and synbodies, can beengineered to bind target molecules very tightly with Kds between 10⁻¹⁰to 10⁻¹² M. In some embodiments, the target neutralization agent bindsthe target molecule with a Kd less than 10⁻⁶, less than 10⁻⁸, less than10⁻⁹, less than 10⁻¹⁰ or less than 10⁻¹² M.

In certain embodiments, the one or more PG pathway inhibitors comprise apolynucleotide that encodes and is adapted to express a PGT inhibitorand/or a PGR inhibitor. In other embodiments, the one or more PG pathwayinhibitors comprise an expression vector that encodes and is adapted toexpress a PGT inhibitor and/or a PGR inhibitor.

In some embodiments, the PG pathway inhibitor is an engineered proteincontaining a TALE sequence or and engineered zinc finger directed at thegene encoding any component of the PG pathway. This TALE or zinc fingercould be designed to bind directly to the gene and inhibit itsexpression by cleaving the gene, altering its nucleotide sequence, ortethering a repressor protein to the gene which serves to silence thegene.

In some embodiments, the PG pathway inhibitor is produced using theCRISPR/CAS system. In this strategy a guide molecule specific for thegene sequence of each pg pathway gene is designed and introduced intothe cell or tissue using delivery systems described above (viruses,plasmids, etc). The action of the CRISPR/CAS system would modify the DNAsequence of the gene such that the PG pathway gene is deleted orinhibited in ability to express the RNA.

In some embodiments, the PG pathway inhibitor is capable of turning offthe transcription of one or more PG pathway genes by targeting thechromatin associated enzymes which post translationally modify histonesin chromatin. Examples of such enzymes are (but not limited to) histonedeacetylases, histone demethylases, histone acetyltransferases, histonemethyltransferases, and helicases.

In some embodiments, the PG pathway inhibitor targets the gene encodingeach component by altering the DNA methylation status of the gene.Compounds which target the TET family of DNA demethylases and the DNAmethyltransferases (DNMT1, DNMTa and DNMTb) could change the expressionof RNA from the any of the genes in the PG pathway.

The expression vector of the present application comprises apolynucleotide encoding a PG pathway inhibitor or a portion thereof. Theexpression vectors also include one or more regulatory sequencesoperably linked to the polynucleotide being expressed. These regulatorysequences are selected based on the type of host cells. It will beappreciated by those skilled in the art that the design of theexpression vector depends on such factors as the choice of the hostcells and the desired expression levels.

In some embodiments, the expression vector is a plasmid vector. In otherembodiments, the expression vector is a viral vector. Examples of viralvectors include, but are not limited to, retrovirus, lentivirus,adenovirus, adeno-associated virus (AAV), herpes virus, or alphavirusvectors. The viral vectors can also be astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, or togavirus vectors. When used in mammalian cells, theexpression vector's control functions are often provided by viralregulatory elements. For example, commonly used promoters are derivedfrom polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40. In someembodiments, the expression vector contains tissue-specific regulatoryelements. Delivery of the expression vector include, but are not limitedto, direct infection with viral vectors, exposing target tissue topolycationic condensed DNA linked or unlinked to killed virus, ligandlinked DNA, gene guns, ionizing radiation, nucleic chargeneutralization, or fusion with cell membranes. Naked plasmid or viralDNA can also be employed. Uptake efficiency may be improved usingbiodegradable latex beads. This method can be further improved bytreating the beads to increase their hydrophobicity. Liposome-basedmethods can also be used to introduce plasmid or viral vector into thetarget tissue.

In some embodiments, the pharmaceutical composition further comprisesone or more active ingredients selected from the group consisting ofanalgesic agents, antimuscarinic agents, antidiuretics, spasmolytics,inhibitors of phosphodiesterase type 5 (PDE 5 inhibitors) and zolpedim.

Examples of antimuscarinic agents include, but are not limited to,oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine,trospium, atropine, and tricyclic antidepressants. Examples ofantidiuretics include, but are not limited to, antidiuretic hormone(ADH), angiotensin II, aldosterone, vasopressin, vasopressin analogs(e.g., desmopressin argipressin, lypressin, felypressin, ornipressin,terlipressin), vasopressin receptor agonists, atrial natriuretic peptide(ANP) and C-type natriuretic peptide (CNP) receptor (i.e., NPR1, NPR2,and NPR3) antagonists (e.g., HS-142-1, isatin, [Asu7,23′]b-ANP-(7-28)],anantin, a cyclic peptide from Streptomyces coerulescens, and 3G12monoclonal antibody), somatostatin type 2 receptor antagonists (e.g.,somatostatin), pharmaceutically-acceptable derivatives, and analogs,salts, hydrates, and solvates thereof. Examples of spasmolytics include,but are not limited to, carisoprodol, benzodiazepines, baclofen,cyclobenzaprine, metaxalone, methocarbamol, clonidine, clonidine analog,and dantrolene. Examples of PDE 5 inhibitors include, but are notlimited to, tadalafil, sildenafil and vardenafil.

The pharmaceutical composition may be formulated for immediate-release,extended-release, delayed-release, or combinations thereof

In some embodiments, the pharmaceutical composition is formulated forimmediate-release.

In other embodiments, the pharmaceutical composition is formulated forextended-release by embedding the active ingredient in a matrix ofinsoluble substance(s) such as acrylics or chitin. The extended-releaseform is designed to release the active ingredient at a predeterminedrate by maintaining a constant drug level for a specific period of time.This can be achieved through a variety of formulations, including, butnot limited to, liposomes and drug-polymer conjugates, such ashydrogels.

An extended-release formulation can be designed to release the activeingredient at a predetermined rate so as to maintain a constant druglevel for a specified, extended period of time, such as up to about 24hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours,about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about3 hours or about 2 hours following administration or following a lagperiod associated with delayed-release of the active ingredient. Theconstant active ingredient level may be maintained by a continuousrelease of the active ingredient or pulsed-release of the activeingredient.

In certain embodiments, the active ingredient in an extended-releaseformulation is released over a time interval of between about 1 to about24 hours, or between 2 to about 12 hours. Alternatively, the activeingredient may be released over about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10 hours, about 11 hours, about 12hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours,about 22 hours or about 24 hours. In yet other embodiments, the activeingredient in an extended-release formulation is released over a timeperiod between about 5 to about 8 hours following administration.

In some embodiments, the extended-release formulation comprises anactive core comprised of one or more inert particles, each in the formof a bead, pellet, pill, granular particle, microcapsule, microsphere,microgranule, nanocapsule, or nanosphere coated on its surfaces withdrugs in the form of a drug-containing coating or film-formingcomposition using, for example, fluid bed techniques or othermethodologies known to those of skill in the art. The inert particle canbe of various sizes, so long as it is large enough to remain poorlydissolved. Alternatively, the active core may be prepared by granulatingand milling and/or by extrusion and spheronization of a polymercomposition containing the drug substance. As used herein, the term“drug” refers to the active ingredient of a pharmaceutical composition.

The active ingredient may be introduced to the inert carrier bytechniques known to one skilled in the art, such as drug layering,powder coating, extrusion/spheronization, roller compaction orgranulation. The amount of active ingredient in the core will depend onthe dose that is required and typically varies from about 1 to 100weight %, about 5 to 100 weight %, about 10 to 100 weight %, about 20 to100 weight %, about 30 to 100 weight %, about 40 to 100 weight %, about50 to 100 weight %, about 60 to 100 weight %, about 70 to 100 weight %,or about 80 to 100 weight %.

Generally, the polymeric coating on the active core will be from about 1to 50% based on the weight of the coated particle, depending on the lagtime required and/or the polymers and coating solvents chosen. Thoseskilled in the art will be able to select an appropriate amount of drugfor coating onto or incorporating into the core to achieve the desireddosage. In one embodiment, the inactive core may be a sugar sphere or abuffer crystal or an encapsulated buffer crystal such as calciumcarbonate, sodium bicarbonate, fumaric acid, tartaric acid, etc. whichalters the microenvironment of the drug to facilitate its release.

Extended-release formulations may utilize a variety of extended-releasecoatings or mechanisms facilitating the gradual release of active agentsover time. In some embodiments, the extended-release agent comprises apolymer controlling release by dissolution controlled release. In aparticular embodiment, the active agent(s) is incorporated in a matrixcomprising an insoluble polymer and drug particles or granules coatedwith polymeric materials of varying thickness. The polymeric materialmay comprise a lipid barrier comprising a waxy material, such ascarnauba wax, beeswax, spermaceti wax, candellila wax, shallac wax,cocoa butter, cetostearyl alcohol, partially hydrogenated vegetableoils, ceresin, paraffin wax, ceresine, myristyl alcohol, stearylalcohol, cetyl alcohol, and stearic acid, along with surfactants, suchas polyoxyethylene sorbitan monooleate. When contacted with an aqueousmedium, such as biological fluids, the polymer coating emulsifies orerodes after a predetermined lag-time depending on the thickness of thepolymer coating. The lag time is independent of gastrointestinalmotility, pH, or gastric residence.

In other embodiments, the extended-release agent comprises a polymericmatrix effecting diffusion controlled release. The matrix may compriseone or more hydrophilic and/or water-swellable, matrix forming polymers,pH-dependent polymers and/or pH-independent polymers.

In one embodiment, the extended-release formulation comprises a watersoluble or water-swellable matrix-forming polymer, optionally containingone or more solubility-enhancing agents and/or release-promoting agents.Upon solubilization of the water soluble polymer, the active agent(s)dissolves (if soluble) and gradually diffuses through the hydratedportion of the matrix. The gel layer grows with time as more waterpermeates into the core of the matrix, increasing the thickness of thegel layer and providing a diffusion barrier to drug release. As theouter layer becomes fully hydrated, the polymer chains become completelyrelaxed and can no longer maintain the integrity of the gel layer,leading to disentanglement and erosion of the outer hydrated polymer onthe surface of the matrix. Water continues to penetrate towards the corethrough the gel layer, until it has been completely eroded. Whereassoluble drugs are released by this combination of diffusion and erosionmechanisms, erosion is the predominant mechanism for insoluble drugs,regardless of dose.

Similarly, water-swellable polymers typically hydrate and swell inbiological fluids forming a homogenous matrix structure that maintainsits shape during drug release and serves as a carrier for the drug,solubility enhancers and/or release promoters. The initial matrixpolymer hydration phase results in slow-release of the drug (lag phase).Once the water swellable polymer is fully hydrated and swollen, waterwithin the matrix can similarly dissolve the drug substance and allowfor its diffusion out through the matrix coating.

Additionally, the porosity of the matrix can be increased due to theleaching out of pH-dependent release promoters so as to release the drugat a faster rate. The rate of the drug release then becomes constant andis a function of drug diffusion through the hydrated polymer gel. Therelease rate from the matrix is dependent upon various factors,including polymer type and level, drug solubility and dose, polymer todrug ratio, filler type and level, polymer to filler ratio, particlesize of drug and polymer, and porosity and shape of the matrix.

Exemplary hydrophilic and/or water-swellable, matrix forming polymersinclude, but are not limited to, cellulosic polymers includinghydroxyalkyl celluloses and carboxyalkyl celluloses such ashydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),hydroxyethylcellulose (HEC), methylcellulose (MC),carboxymethylcellulose (CMC); powdered cellulose such asmicrocrystalline cellulose, cellulose acetate, ethylcellulose, saltsthereof, and combinations thereof; alginates; gums includingheteropolysaccharide gums and homopolysaccharide gums such as xanthan,tragacanth, pectin, acacia, karaya, alginates, agar, guar, hydroxypropylguar, veegum, carrageenan, locust bean gum, gellan gum, and derivativestherefrom; acrylic resins including polymers and copolymers of acrylicacid, methacrylic acid, methyl acrylate, and methyl methacrylate; andcross-linked polyacrylic acid derivatives such as Carbomers (e.g.,CARBOPOL®, including CARBOPOL° 71G NF, which is available in variousmolecular weight grades from Noveon, Inc., Cincinnati, Ohio),carageenan; polyvinyl acetate (e.g., KOLLIDON® SR); and polyvinylpyrrolidone and its derivatives such as crospovidone, polyethyleneoxides, and polyvinyl alcohol. Preferred hydrophilic and water-swellablepolymers include the cellulosic polymers, especially HPMC.

The extended-release formulation may further comprise at least onebinder that is capable of cross-linking the hydrophilic compound to forma hydrophilic polymer matrix (e.g., a gel matrix) in an aqueous medium,including biological fluids.

Exemplary binders include homopolysaccharides such as galactomannangums, guar gum, hydroxypropyl guar gum, hydroxypropylcellulose (HPC;e.g., Klucel EXF), and locust bean gum. In other embodiments, the binderis an alginic acid derivative, HPC or microcrystallized cellulose (MCC).Other binders include, but are not limited to, starches,microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropylmethyl cellulose, and polyvinylpyrrolidone.

In one embodiment, the introduction method is drug layering by sprayinga suspension of active agent(s) and a binder onto the inert carrier.

The binder may be present in the bead formulation in an amount of about0.1% to about 15% by weight and preferably of from about 0.2% to about10% by weight.

In some embodiments, the hydrophilic polymer matrix may further includean ionic polymer, a non-ionic polymer, or water-insoluble hydrophobicpolymer to provide a stronger gel layer and/or reduce pore quantity anddimensions in the matrix so as to slow diffusion and erosion rates andconcomitant release of the active agent(s). This may additionallysuppress the initial burst effect and produce a more steady “zero orderrelease” of active agent(s).

Exemplary ionic polymers for slowing dissolution rate include bothanionic and cationic polymers. Exemplary anionic polymers include, forexample, sodium carboxymethylcellulose (Na CMC); sodium alginate,polymers of acrylic acid or carbomers (e.g., CARBOPOL° 934, 940, 974PNF); enteric polymers such as polyvinyl acetate phthalate (PVAP),methacrylic acid copolymers (e.g., EUDRAGIT° L100, L 30D 55, A, and FS30D), and hypromellose acetate succinate (AQUAT HPMCAS); and xanthangum. Exemplary cationic polymers include, for example,dimethylaminoethyl methacrylate copolymer (e.g., EUDRAGIT° E 100).Incorporation of anionic polymers, particularly enteric polymers, isuseful for developing a pH-independent release profile for weakly basicdrugs as compared to hydrophilic polymer alone.

Exemplary non-ionic polymers for slowing dissolution rate, include, forexample, hydroxypropylcellulose (HPC) and polyethylene oxide (PEO)(e.g., POLYOX™)

Exemplary hydrophobic polymers include ethylcellulose (e.g., ETHOCEL™)SURELEASE®), cellulose acetate, methacrylic acid copolymers (e.g.,EUDRAGIT® NE 30D), ammonio-methacrylate copolymers (e.g., EUDRAGIT® RL100 or PO RS100), polyvinyl acetate, glyceryl monostearate, fatty acidssuch as acetyl tributyl citrate, and combinations and derivativesthereof.

The swellable polymer can be incorporated in the formulation inproportion from 1% to 50% by weight, preferably from 5% to 40% byweight, most preferably from 5% to 20% by weight. The swellable polymersand binders may be incorporated in the formulation either prior to orafter granulation. The polymers can also be dispersed in organicsolvents or hydro-alcohols and sprayed during granulation.

Exemplary release-promoting agents include pH-dependent enteric polymersthat remain intact at a pH value lower than about 4.0 and dissolve at pHvalues higher than 4.0, preferably higher than 5.0, most preferablyabout 6.0, and are considered useful as release-promoting agents forthis invention. Exemplary pH-dependent polymers include, but are notlimited to, methacarylic acid copolymers; methacrylic acid-methylmethacrylate copolymers (e.g., EUDRAGIT® L100 (Type A), EUDRAGIT® S100(Type B), Rohm GmbH, Germany), methacrylic acid-ethyl acrylatecopolymers (e.g., EUDRAGIT° L100-55 (Type C) and EUDRAGIT° L30D-55copolymer dispersion, Rohm GmbH, Germany); copolymers of methacrylicacid-methyl methacrylate and methyl methacrylate (EUDRAGIT® FS);terpolymers of methacrylic acid, methacrylate, and ethyl acrylate,cellulose acetate phthalates (CAP); hydroxypropyl methylcellulosephthalate (HPMCP) (e.g., HP-55, HP-50, HP-555, Shinetsu Chemical,Japan); polyvinyl acetate phthalates (PVAP) (e.g., COATERIC®, OPADRY®enteric white OY-P-7171); polyvinylbutyrate acetate, cellulose acetatesuccinates (CAS); hydroxypropyl methylcellulose acetate succinate(HPMCAS) (e.g., HPMCAS LF Grade, MF Grade, and HF Grade, includingAQOAT® LF and AQOAT° MF, Shin-Etsu Chemical, Japan), shellac (e.g.,MARCOATTM 125 and MARCOAT™ 125N); vinyl acetate-maleic anhydridecopolymer, styrene-maleic monoester copolymer, carboxymethylethylcellulose (CMEC, Freund Corporation, Japan); cellulose acetatephthalates (CAP) (e.g., AQUATERIC®), cellulose acetate trimellitates(CAT), and mixtures of two or more thereof at weight ratios betweenabout 2:1 to about 5:1, such as a mixture of EUDRAGIT® L 100-55 andEUDRAGIT® S 100 at a weight ratio of about 3:1 to about 2:1, or amixture of EUDRAGIT® L 30 D-55 and EUIDRAGIT® FS at a weight ratio ofabout 3:1 to about 5:1.

These polymers may be used either alone or in combination, or togetherwith polymers other than those mentioned above. Preferred entericpH-dependent polymers are the pharmaceutically acceptable methacrylicacid copolymers. These copolymers are anionic polymers based onmethacrylic acid and methyl methacrylate and, preferably, have a meanmolecular weight of about 50,000 to 200,000, preferably about 135,000. Aratio of free carboxyl groups to methyl-esterified carboxyl groups inthese copolymers may range, for example, from 1:1 to 1:3, e.g. around1:1 or 1:2. The release promoters are not limited to pH dependentpolymers. Other hydrophilic molecules that dissolve rapidly and leachout of the dosage form quickly leaving a porous structure can be also beused for the same purpose.

In some embodiments, the matrix may include a combination of releasepromoters and solubility enhancing agents. The solubility enhancingagents can be ionic and non-ionic surfactants, complexing agents,hydrophilic polymers, and pH modifiers such as acidifying agents andalkalinizing agents, as well as molecules that increase the solubilityof poorly soluble drug through molecular entrapment. Several solubilityenhancing agents can be utilized simultaneously.

Solubility enhancing agents may include surface active agents, such assodium docusate; sodium lauryl sulfate; sodium stearyl fumarate; Tweens®and Spans (PEO modified sorbitan monoesters and fatty acid sorbitanesters); poly(ethylene oxide)-polypropylene oxide-poly(ethylene oxide)block copolymers (aka PLURONICS™); complexing agents such as lowmolecular weight polyvinyl pyrrolidone and low molecular weighthydroxypropyl methyl cellulose; molecules that aid solubility bymolecular entrapment such as cyclodextrins and pH modifying agents,including acidifying agents such as citric acid, fumaric acid, tartaricacid, and hydrochloric acid, and alkalizing agents such as meglumine andsodium hydroxide.

Solubility enhancing agents typically constitute from 1% to 80% byweight, from 1% to 60% by weight, from 1% to 50% by weight, from 1% to40% by weight and from 1% to 30% by weight, of the dosage form and canbe incorporated in a variety of ways. They can be incorporated in theformulation prior to granulation in dry or wet form. They can also beadded to the formulation after the rest of the materials are granulatedor otherwise processed. During granulation, solubility enhancing agentscan be sprayed as solutions with or without a binder.

In one embodiment, the extended-release formulation comprises awater-insoluble water-permeable polymeric coating or matrix comprisingone or more water-insoluble water-permeable film-forming over the activecore. The coating may additionally include one or more water solublepolymers and/or one or more plasticizers. The water-insoluble polymercoating comprises a barrier coating for release of active agents in thecore, wherein lower molecular weight (viscosity) grades exhibit fasterrelease rates as compared to higher viscosity grades.

In some embodiments, the water-insoluble film-forming polymers includeone or more alkyl cellulose ethers, such as ethyl celluloses andmixtures thereof, (e.g., ethyl cellulose grades PR100, PR45, PR20, PR10,and PR7; ETHOCEL®, Dow).

In some embodiments, the water-insoluble polymer provides suitableproperties (e.g., extended-release characteristics, mechanicalproperties, and coating properties) without the need for a plasticizer.For example, coatings comprising polyvinyl acetate (PVA), neutralcopolymers of acrylate/methacrylate esters such as commerciallyavailable Eudragit NE3OD from Evonik Industries, ethyl cellulose incombination with hydroxypropylcellulose, waxes, etc. can be appliedwithout plasticizers.

In yet another embodiment, the water-insoluble polymer matrix mayfurther include a plasticizer. The amount of plasticizer requireddepends upon the plasticizer, the properties of the water-insolublepolymer and the ultimate desired properties of the coating. Suitablelevels of plasticizer range from about 1% to about 20%, from about 3% toabout 20%, about 3% to about 5%, about 7% to about 10%, about 12% toabout 15%, about 17% to about 20%, or about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 15%, or about 20% by weight relative to the total weight of thecoating, inclusive of all ranges and sub-ranges therebetween.

Exemplary plasticizers include, but are not limited to, triacetin,acetylated monoglyceride, oils (castor oil, hydrogenated castor oil,grape seed oil, sesame oil, olive oil, and etc.), citrate esters,triethyl citrate, acetyltriethyl citrate acetyltributyl citrate,tributyl citrate, acetyl tri-n-butyl citrate, diethyl phthalate, dibutylphthalate, dioctyl phthalate, methyl paraben, propyl paraben, propylparaben, butyl paraben, diethyl sebacate, dibutyl sebacate,glyceroltributyrate, substituted triglycerides and glycerides,monoacetylated and diacetylated glycerides (e.g., MYVACET® 9-45),glyceryl monostearate, glycerol tributyrate, polysorbate 80,polyethyleneglycol (such as PEG-4000 and PEG-400), propyleneglycol,1,2-propyleneglycol, glycerin, sorbitol, diethyl oxalate, diethylmalate, diethyl fumarate, diethylmalonate, dibutyl succinate, fattyacids, glycerin, sorbitol, diethyl oxalate, diethyl malate, diethylmaleate, diethyl fumarate, diethyl succinate, diethyl malonate, dioctylphthalate, dibutyl sebacate, and mixtures thereof. The plasticizer canhave surfactant properties, such that it can act as a release modifier.For example, non-ionic detergents such as Brij 58 (polyoxyethylene (20)cetyl ether), and the like, can be used.

Plasticizers can be high boiling point organic solvents used to impartflexibility to otherwise hard or brittle polymeric materials and canaffect the release profile for the active agent(s). Plasticizersgenerally cause a reduction in the cohesive intermolecular forces alongthe polymer chains resulting in various changes in polymer properties.These changes include, but are not limited to, a reduction in tensilestrength and increase in elongation and a reduction in the glasstransition or softening temperature of the polymer. The amount andchoice of the plasticizer can affect the hardness of a tablet, forexample, and can even affect its dissolution or disintegrationcharacteristics, as well as its physical and chemical stability. Certainplasticizers can increase the elasticity and/or pliability of a coat,thereby decreasing the coat's brittleness.

In another embodiment, the extended-release formulation comprises acombination of at least two gel-forming polymers, including at least onenon-ionic gel-forming polymer and/or at least one anionic gel-formingpolymer. The gel formed by the combination of gel-forming polymersprovides controlled release, such that when the formulation is ingestedand comes into contact with the gastrointestinal fluids, the polymersnearest the surface hydrate to form a viscous gel layer. Due to the highviscosity, the viscous layer dissolves away only gradually, exposing thematerial below to the same process. The mass thus dissolves away slowly,thereby slowly releasing the active ingredient into the gastrointestinalfluids. The combination of at least two gel-forming polymers enablesproperties of the resultant gel, such as viscosity, to be manipulated inorder to provide the desired release profile.

In a particular embodiment, the formulation comprises at least onenon-ionic gel-forming polymer and at least one anionic gel-formingpolymer. In another embodiment, the formulation comprises two differentnon-ionic gel-forming polymers. In yet another embodiment, theformulation comprises a combination of non-ionic gel-forming polymerswith the same chemistry, but different solubilities, viscosities, and/ormolecular weights (for example, a combination of hydroxyproplylmethylcellulose of different viscosity grades, such as HPMC K100 andHPMC K15M or HPMC K100M).

Exemplary anionic gel forming polymers include, but are not limited to,sodium carboxymethylcellulose (Na CMC), carboxymethyl cellulose (CMC),anionic polysaccharides such as sodium alginate, alginic acid, pectin,polyglucuronic acid (poly-α- and -β-1,4-glucuronic acid),polygalacturonic acid (pectic acid), chondroitin sulfate, carrageenan,furcellaran, anionic gums such as xanthan gum, polymers of acrylic acidor carbomers (Carbopol® 934, 940, 974P NF), Carbopol® copolymers, aPemulen® polymer, polycarbophil, and others.

Exemplary non-ionic gel-forming polymers include, but are not limitedto, Povidone (PVP: polyvinyl pyrrolidone), polyvinyl alcohol, copolymerof PVP and polyvinyl acetate, HPC (hydroxypropyl cellulose), HPMC(hydroxypropyl methylcellulose), hydroxyethyl cellulose, hydroxymethylcellulose, gelatin, polyethylene oxide, acacia, dextrin, starch,polyhydroxyethylmethacrylate (PHEMA), water soluble nonionicpolymethacrylates and their copolymers, modified cellulose, modifiedpolysaccharides, nonionic gums, nonionic polysaccharides, and/ormixtures thereof.

The formulation may optionally comprise an enteric polymer as describedabove and/or at least one excipient, such as a filler, a binder (asdescribed above), a disintegrant and/or a flow aid or glidant.

Exemplary fillers include, but are not limited to, lactose, glucose,fructose, sucrose, dicalcium phosphate, sugar alcohols also known as“sugar polyol” such as sorbitol, manitol, lactitol, xylitol, isomalt,erythritol, and hydrogenated starch hydrolysates (a blend of severalsugar alcohols), corn starch, potato starch, sodiumcarboxymethycellulose, ethylcellulose and cellulose acetate, entericpolymers, or a mixture thereof.

Exemplary binders include, but are not limited to, water-solublehydrophilic polymers such as Povidone (PVP: polyvinyl pyrrolidone),copovidone (a copolymer of polyvinyl pyrrolidone and polyvinyl acetate),low molecular weight HPC (hydroxypropyl cellulose), low molecular weightHPMC (hydroxypropyl methylcellulose), low molecular weight carboxymethyl cellulose, ethylcellulose, gelatin, polyethylene oxide, acacia,dextrin, magnesium aluminum silicate, and starch and polymethacrylatessuch as Eudragit NE 30D, Eudragit RL, Eudragit RS, Eudragit E, polyvinylacetate, enteric polymers, or mixtures thereof.

Exemplary disintegrants include, but are not limited to, low-substitutedcarboxymethyl cellulose sodium, crospovidone (cross-linked polyvinylpyrrolidone), sodium carboxymethyl starch (sodium starch glycolate),cross-linked sodium carboxymethyl cellulose (Croscarmellose),pregelatinized starch (starch 1500), microcrystalline cellulose, waterinsoluble starch, calcium carboxymethyl cellulose, low substitutedhydroxypropyl cellulose, and magnesium or aluminum silicate.

Exemplary glidants include but are not limited to, magnesium, silicondioxide, talc, starch, titanium dioxide, and the like.

In yet another embodiment, the extended-release formulation is formed bycoating a water soluble/dispersible drug-containing particle, such as abead or bead population therein (as described above), with a coatingmaterial and, optionally, a pore former and other excipients. Thecoating material is preferably selected from a group comprisingcellulosic polymers such as ethylcellulose (e.g., SURELEASE®),methylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose,cellulose acetate, and cellulose acetate phthalate; polyvinyl alcohol;acrylic polymers such as polyacrylates, polymethacrylates, andcopolymers thereof; and other water-based or solvent-based coatingmaterials. The release-controlling coating for a given bead populationmay be controlled by at least one parameter of the release controllingcoating, such as the nature of the coating, coating level, type andconcentration of a pore former, process parameters, and combinationsthereof. Thus, changing a parameter, such as a pore formerconcentration, or the conditions of the curing, allows for changes inthe release of active agent(s) from any given bead population, therebyallowing for selective adjustment of the formulation to a pre-determinedrelease profile.

Pore formers suitable for use in the release controlling coating hereincan be organic or inorganic agents and include materials that can bedissolved, extracted or leached from the coating in the environment ofuse. Exemplary pore forming agents include, but are not limited to,organic compounds such as mono-, oligo-, and polysaccharides includingsucrose, glucose, fructose, mannitol, mannose, galactose, sorbitol,pullulan, and dextran; polymers soluble in the environment of use suchas water-soluble hydrophilic polymers, hydroxyalkylcelluloses,carboxyalkylcelluloses, hydroxypropylmethylcellulose, cellulose ethers,acrylic resins, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone,polyethylene oxide, Carbowaxes, Carbopol, and the like, diols, polyols,polyhydric alcohols, polyalkylene glycols, polyethylene glycols,polypropylene glycols, or block polymers thereof, polyglycols, andpoly(α-Ω)alkylenediols; and inorganic compounds such as alkali metalsalts, lithium carbonate, sodium chloride, sodium bromide, potassiumchloride, potassium sulfate, potassium phosphate, sodium acetate, sodiumcitrate, suitable calcium salts, combination thereof, and the like.

The release controlling coating can further comprise other additivesknown in the art, such as plasticizers, anti-adherents, glidants (orflow aids) and antifoams. In some embodiments, the coated particles orbeads may additionally include an “overcoat,” to provide, for example,moisture protection, static charge reduction, taste-masking, flavoring,coloring, and/or polish or other cosmetic appeal to the beads. Suitablecoating materials for such an overcoat are known in the art and include,but are not limited to, cellulosic polymers such ashydroxypropylmethylcellulose, hydroxypropylcellulose, andmicrocrystalline cellulose or combinations thereof (for example, variousOPADRY® coating materials).

The coated particles or beads may additionally contain enhancers thatmay be exemplified by, but not limited to, solubility enhancers,dissolution enhancers, absorption enhancers, permeability enhancers,stabilizers, complexing agents, enzyme inhibitors, p-glycoproteininhibitors, and multidrug resistance protein inhibitors. Alternatively,the formulation can also contain enhancers that are separated from thecoated particles, for example, in a separate population of beads or as apowder. In yet another embodiment, the enhancer(s) may be contained in aseparate layer on coated particles either under or above the releasecontrolling coating.

In other embodiments, the extended-release formulation is formulated torelease the active agent(s) by an osmotic mechanism. By way of example,a capsule may be formulated with a single osmotic unit or it mayincorporate 2, 3, 4, 5, or 6 push-pull units encapsulated within a hardgelatin capsule, whereby each bilayer push pull unit contains an osmoticpush layer and a drug layer, both surrounded by a semi-permeablemembrane. One or more orifices are drilled through the membrane next tothe drug layer. This membrane may be additionally covered with apH-dependent enteric coating to prevent release until after gastricemptying. The gelatin capsule dissolves immediately after ingestion. Asthe push pull unit(s) enters the small intestine, the enteric coatingbreaks down, which then allows fluid to flow through the semi-permeablemembrane, swelling the osmotic push compartment to force drugs outthrough the orifice(s) at a rate precisely controlled by the rate ofwater transport through the semi-permeable membrane. Release of drugscan occur over a constant rate for up to 24 hours or more.

The osmotic push layer comprises one or more osmotic agents creating thedriving force for transport of water through the semi-permeable membraneinto the core of the delivery vehicle. One class of osmotic agentsincludes water-swellable hydrophilic polymers, also referred to as“osmopolymers” and “hydrogels,” including, but not limited to,hydrophilic vinyl and acrylic polymers, polysaccharides such as calciumalginate, polyethylene oxide (PEO), polyethylene glycol (PEG),polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate),poly(acrylic) acid, poly(methacrylic) acid, polyvinylpyrrolidone (PVP),crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVPcopolymers with hydrophobic monomers such as methyl methacrylate andvinyl acetate, hydrophilic polyurethanes containing large PEO blocks,sodium croscarmellose, carrageenan, hydroxyethyl cellulose (HEC),hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC),carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodiumalginate, polycarbophil, gelatin, xanthan gum, and sodium starchglycolate.

Another class of osmotic agents includes osmogens, which are capable ofimbibing water to effect an osmotic pressure gradient across thesemi-permeable membrane. Exemplary osmogens include, but are not limitedto, inorganic salts such as magnesium sulfate, magnesium chloride,calcium chloride, sodium chloride, lithium chloride, potassium sulfate,potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate,potassium chloride, and sodium sulfate; sugars such as dextrose,fructose, glucose, inositol, lactose, maltose, mannitol, raffinose,sorbitol, sucrose, trehalose, and xylitol; organic acids such asascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid,sebacic acid, sorbic acid, adipic acid, edetic acid, glutamic acid,p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; andmixtures thereof.

Materials useful in forming the semipermeable membrane include variousgrades of acrylics, vinyls, ethers, polyamides, polyesters, andcellulosic derivatives that are water-permeable and water-insoluble atphysiologically relevant pHs, or are susceptible to being renderedwater-insoluble by chemical alteration, such as crosslinking.

In some embodiments, the extended-release formulation comprises apolysaccharide coating that is resistant to erosion in both the stomachand intestine. Such polymers can be only degraded in the colon, whichcontains a large microflora containing biodegradable enzymes breakingdown, for example, the polysaccharide coatings to release the drugcontents in a controlled, time-dependent manner. Exemplarypolysaccharide coatings may include, for example, amylose,arabinogalactan, chitosan, chondroitin sulfate, cyclodextrin, dextran,guar gum, pectin, xylan, and combinations or derivatives therefrom.

In some embodiments, the pharmaceutical composition is formulated fordelayed-release or delayed-extended-release. In some embodiments, thedelayed extended-release formulation includes an extended-releaseformulation coated with an enteric coating, which is a barrier appliedto oral medication that prevents release of medication before it reachesthe small intestine. Delayed-release formulations, such as entericcoatings, prevent drugs having an irritant effect on the stomach, suchas aspirin, from dissolving in the stomach. As used herein, the term“enteric coating” is a coating comprising of one or more polymers havinga pH dependent or pH-independent release profile. An enteric coated pillwill not dissolve in the acidic juices of the stomach (pH˜3), but theywill in the alkaline (pH 7-9) environment present in the small intestineor colon. An enteric polymer coating typically resists releases of theactive agents until sometime after a gastric emptying lag period ofabout 3-4 hours after administration. Accordingly, a formulation thatreleases it component “after gastric emptying” refers to a delayedformulation that releases the active ingredient(s) after the formulationis emptied from the stomach and enters intestine.

Such coatings are also used to protect acid-unstable drugs from thestomach's acidic exposure, delivering them instead to a basic pHenvironment (intestine's pH 5.5 and above) where they do not degrade andgive their desired action. The term “pulsatile-release” is a type ofdelayed-release, which is used herein with reference to a drugformulation that provides rapid and transient release of the drug withina short time period immediately after a predetermined lag period,thereby producing a “pulsed” plasma profile of the drug after drugadministration. Formulations may be designed to provide a singlepulsatile release or multiple pulsatile releases at predetermined timeintervals following administration, or a pulsatile release (e.g., 20-60%of the active ingredient) followed with extended release over a periodof time (e.g., a continuous release of the remainder of the activeingredient). A delayed-release or pulsatile release formulationgenerally comprises one or more elements covered with a barrier coating,which dissolves, erodes or ruptures following a specified lag phase.

A barrier coating for delayed-release may consist of a variety ofdifferent materials, depending on the objective. In addition, aformulation may comprise a plurality of barrier coatings to facilitaterelease in a temporal manner. The coating may be a sugar coating, a filmcoating (e.g., based on hydroxypropyl methylcellulose, methylcellulose,methyl hydroxyethylcellulose, hydroxypropylcellulose,carboxymethylcellulose, acrylate copolymers, polyethylene glycols,and/or polyvinylpyrrolidone) or a coating based on methacrylic acidcopolymer, cellulose acetate phthalate, hydroxypropyl methylcellulosephthalate, hydroxypropyl methylcellulose acetate succinate, polyvinylacetate phthalate, shellac, and/or ethylcellulose. Furthermore, theformulation may additionally include a time delay material such asglyceryl monostearate or glyceryl distearate.

In some embodiments, the delayed-extended-release formulation includesan enteric coating comprising one or more polymers facilitating releaseof active agents in proximal or distal regions of the gastrointestinaltract. pH dependent enteric coatings comprise one or more pH-dependentor pH-sensitive polymers that maintain their structural integrity at lowpH, as in the stomach, but dissolve in higher pH environments in moredistal regions of the gastrointestinal tract, such as the smallintestine, where the drug contents are released. For purposes of thepresent invention, “pH dependent” is defined as having characteristics(e.g., dissolution) which vary according to environmental pH. ExemplarypH-dependent polymers have been described earlier. pH-dependent polymerstypically exhibit a characteristic pH optimum for dissolution. In someembodiments, the pH-dependent polymer exhibits a pH optimum betweenabout 5.0 and 5.5, between about 5.5 and 6.0, between about 6.0 and 6.5,or between about 6.5 and 7.0. In other embodiments, the pH-dependentpolymer exhibits a pH optimum of ≥5.0, of ≥5.5, of ≥6.0, of ≥6.5, or of≥7.0.

In certain embodiments, the coating methodology employs the blending ofone or more pH-dependent and one or more pH-independent polymers. Theblending of pH-dependent and pH-independent polymers can reduce therelease rate of active ingredients once the soluble polymer has reachedits optimum pH of solubilization.

In some embodiments, a “delayed-release” or “delayed-extended-release”profile can be obtained using a water insoluble capsule body containingone or more active agents, wherein the capsule body closed at one endwith an insoluble, but permeable and swellable hydrogel plug. Uponcontact with gastrointestinal fluid or dissolution medium, the plugswells, pushing itself out of the capsule and releasing the drugs aftera pre-determined lag time, which can be controlled by, for example, theposition and dimensions of the plug. The capsule body may be furthercoated with an outer pH-dependent enteric coating keeping the capsuleintact until it reaches the small intestine. Suitable plug materialsinclude, for example, polymethacrylates, erodible compressed polymers(e.g., HPMC, polyvinyl alcohol), congealed melted polymer (e.g.,glyceryl mono oleate), and enzymatically controlled erodible polymers(e.g., polysaccharides, such as amylose, arabinogalactan, chitosan,chondroitin sulfate, cyclodextrin, dextran, guar gum, pectin and xylan).

In other embodiments, capsules or bilayered tablets may be formulated tocontain a drug-containing core, covered by a swelling layer and an outerinsoluble, but semi-permeable polymer coating or membrane. The lag timeprior to rupture can be controlled by the permeation and mechanicalproperties of the polymer coating and the swelling behavior of theswelling layer. Typically, the swelling layer comprises one or moreswelling agents, such as swellable hydrophilic polymers that swell andretain water in their structures.

Exemplary water swellable materials to be used in the delayed-releasecoating include, but are not limited to, polyethylene oxides (havinge.g., an average molecular weight between 1,000,000 and 7,000,000, suchas POLYYOX®); methylcellulose; hydroxypropyl cellulose; hydroxypropylmethylcellulose; polyalkylene oxides having a weight average molecularweight of 100,000 to 6,000,000, including, but not limited to,poly(methylene oxide), poly(butylene oxide), poly(hydroxy alkylmethacrylate) having a molecular weight of 25,000 to 5,000,000,poly(vinyl)alcohol having a low acetal residue, which is cross-linkedwith glyoxal, formaldehyde, or glutaraldehyde, and having a degree ofpolymerization from 200 to 30,000; mixtures of methyl cellulose,cross-linked agar, and carboxymethyl cellulose; hydrogel formingcopolymers produced by forming a dispersion of a finely dividedcopolymer of maleic anhydride with styrene, ethylene, propylene,butylene, or isobutylene cross-linked with from 0.001 to 0.5 moles ofsaturated cross-linking agent per mole of maleic anyhydride in thecopolymer; CARBOPOL® acidic carboxy polymers having a molecular weightof 450,000 to 4,000,000; CYANAMER® polyacrylamides; cross-linked waterswellable indenemaleicanhydride polymers; GOODRITE® polyacrylic acidhaving a molecular weight of 80,000 to 200,000; starch graft copolymers;AQUA-KEEPS® acrylate polymer polysaccharides composed of condensedglucose units such as diester cross-linked polyglucan; carbomers havinga viscosity of 3,000 to 60,000 mPas as a 0.5%-1% w/v aqueous solution;cellulose ethers such as hydroxypropylcellulose having a viscosity ofabout 1000-7000 mPa s as a 1% w/v aqueous solution (25° C.);hydroxypropyl methylcellulose having a viscosity of about 1000 orhigher, preferably 2,500 or higher to a maximum of 25,000 mPas as a 2%w/v aqueous solution; polyvinylpyrrolidone having a viscosity of about300-700 mPas as a 10% w/v aqueous solution at 20° C.; and combinationsthereof.

Alternatively, the release time of the drugs can be controlled by adisintegration lag time depending on the balance between thetolerability and thickness of a water insoluble polymer membrane (suchas ethyl cellulose, EC) containing predefined micropores at the bottomof the body and the amount of a swellable excipient, such as lowsubstituted hydroxypropyl cellulose (L-HPC) and sodium glycolate. Afteroral administration, GI fluids permeate through the micropores, causingswelling of the swellable excipients, which produces an inner pressuredisengaging the capsular components, including a first capsule bodycontaining the swellable materials, a second capsule body containing thedrugs, and an outer cap attached to the first capsule body.

The delayed-release coating layer may further comprise anti-tackinessagents, such as talc and glyceryl monostearate. The delayed-releasecoating layer may further comprise one or more plasticizers including,but not limited to, triethyl citrate, acetyl triethyl citrate,acetyltributyl citrate, polyethylene glycol acetylated monoglycerides,glycerin, triacetin, propylene glycol, phthalate esters (e.g., diethylphthalate, dibutyl phthalate), titanium dioxide, ferric oxides, castoroil, sorbitol, and dibutyl sebacate.

In another embodiment, the delayed-release formulation employs awater-permeable but insoluble film coating to enclose the activeingredient and an osmotic agent. As water from the gut slowly diffusesthrough the film into the core, the core swells until the film bursts,thereby releasing the active ingredients. The film coating may beadjusted to permit various rates of water permeation or release time.

In another embodiment, the delayed release formulation employs awater-impermeable tablet coating whereby water enters through acontrolled aperture in the coating until the core bursts. When thetablet bursts, the drug contents are released immediately or over alonger period of time. These and other techniques may be modified toallow for a pre-determined lag period before release of drugs isinitiated.

In another embodiment, the active agents are delivered in a formulationto provide both delayed-release and extended-release(delayed-extended-release). The term “delayed-extended-release”is usedherein with reference to a drug formulation providing pulsatile releaseof active agents at a pre-determined time or lag period followingadministration, which is then followed by extended-release of the activeagents thereafter.

In some embodiments, immediate-release, extended-release,delayed-release, or delayed-extended-release formulations comprises anactive core comprised of one or more inert particles, each in the formof a bead, pellet, pill, granular particle, microcapsule, microsphere,microgranule, nanocapsule, or nanosphere coated on its surfaces withdrugs in the form of e.g., a drug-containing film-forming compositionusing, for example, fluid bed techniques or other methodologies known tothose of skill in the art. The inert particle can be of various sizes,so long as it is large enough to remain poorly dissolved. Alternatively,the active core may be prepared by granulating and milling and/or byextrusion and spheronization of a polymer composition containing thedrug substance.

The amount of drug in the core will depend on the dose that is requiredand typically varies from about 1 to 100 weight %, about 5 to 100 weight%, about 10 to 100 weight %, about 20 to 100 weight %, about 30 to 100weight %, about 40 to 100 weight %, about 50 to 100 weight %, about 60to 100 weight %, about 70 to 100 weight %, or about 80 to 100 weight %.Generally, the polymeric coating on the active core will be from about 1to 50% based on the weight of the coated particle, depending on the lagtime and type of release profile required and/or the polymers andcoating solvents chosen. Those skilled in the art will be able to selectan appropriate amount of drug for coating onto or incorporating into thecore to achieve the desired dosage. In one embodiment, the inactive coremay be a sugar sphere or a buffer crystal or an encapsulated buffercrystal such as calcium carbonate, sodium bicarbonate, fumaric acid,tartaric acid, etc. which alters the microenvironment of the drug tofacilitate its release.

In some embodiments, for example, delayed-release ordelayed-extended-release compositions may formed by coating a watersoluble/dispersible drug-containing particle, such as a bead, with amixture of a water insoluble polymer and an enteric polymer, wherein thewater insoluble polymer and the enteric polymer may be present at aweight ratio of from 4:1 to 1:1, and the total weight of the coatings is10 to 60 weight % based on the total weight of the coated beads. Thedrug layered beads may optionally include an inner dissolution ratecontrolling membrane of ethylcellulose. The composition of the outerlayer, as well as the individual weights of the inner and outer layersof the polymeric membrane are optimized for achieving desired circadianrhythm release profiles for a given active, which are predicted based onin vitro/in vivo correlations.

In other embodiments the formulations may comprise a mixture ofimmediate-release drug-containing particles without a dissolution ratecontrolling polymer membrane and delayed-extended-release beadsexhibiting, for example, a lag time of 2-4 hours following oraladministration, thus providing a two-pulse release profile.

In some embodiments, the active core is coated with one or more layersof dissolution rate-controlling polymers to obtain desired releaseprofiles with or without a lag time. An inner layer membrane can largelycontrol the rate of drug release following imbibition of water or bodyfluids into the core, while the outer layer membrane can provide for adesired lag time (the period of no or little drug release followingimbibition of water or body fluids into the core). The inner layermembrane may comprise a water insoluble polymer, or a mixture of waterinsoluble and water soluble polymers.

The polymers suitable for the outer membrane, which largely controls thelag time of up to 6 hours may comprise an enteric polymer, as describedabove, and a water insoluble polymer at 10 to 50 weight %. The ratio ofwater insoluble polymer to enteric polymer may vary from 4:1 to 1:2,preferably the polymers are present at a ratio of about 1:1. The waterinsoluble polymer typically used is ethylcellulose.

Exemplary water insoluble polymers include ethylcellulose, polyvinylacetate (Kollicoat SR#0D from BASF), neutral copolymers based on ethylacrylate and methylmethacrylate, copolymers of acrylic and methacrylicacid esters with quaternary ammonium groups such as EUDRAGIT® NE, RS andRS30D, RL or RL30D, and the like. Exemplary water soluble polymersinclude low molecular weight HPMC, HPC, methylcellulose, polyethyleneglycol (PEG of molecular weight>3000) at a thickness ranging from 1weight % up to 10 weight % depending on the solubility of the active inwater and the solvent or latex suspension based coating formulationused. The water insoluble polymer to water soluble polymer may typicallyvary from 95:5 to 60:40, preferably from 80:20 to 65:35. In someembodiments, AMBERLITE™ IRP69 resin is used as an extended-releasecarrier. AMBERLITE™ IRP69 is an insoluble, strongly acidic, sodium formcation exchange resin that is suitable as carrier for cationic (basic)substances. In other embodiments, DUOLITE™ AP143/1093 resin is used asan extended-release carrier. DUOLITE™ AP143/1093 is an insoluble,strongly basic, anion exchange resin that is suitable as a carrier foranionic (acidic) substances. When used as a drug carrier, AMBERLITE™IRP69 or/and DUOLITE™ AP143/1093 resin provides a means for bindingmedicinal agents onto an insoluble polymeric matrix. Extended-release isachieved through the formation of resin-drug complexes (drug resinates).The drug is released from the resin in vivo as the drug reachesequilibrium with the high electrolyte concentrations, which are typicalof the gastrointestinal tract. More hydrophobic drugs will usually elutefrom the resin at a lower rate, owing to hydrophobic interactions withthe aromatic structure of the cation exchange system.

In some embodiments, the pharmaceutical composition is formulated fororal administration. Oral dosage forms include, for example, tablets,capsules, and caplets and may also comprise a plurality of granules,beads, powders, or pellets that may or may not be encapsulated. Tabletsand capsules represent the most convenient oral dosage forms, in whichcase solid pharmaceutical carriers are employed.

In a delayed-release formulation, one or more barrier coatings may beapplied to pellets, tablets, or capsules to facilitate slow dissolutionand concomitant release of drugs into the intestine. Typically, thebarrier coating contains one or more polymers encasing, surrounding, orforming a layer, or membrane around the therapeutic composition oractive core. In some embodiments, the active agents are delivered in aformulation to provide delayed-release at a pre-determined timefollowing administration. The delay may be up to about 10 minutes, about20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, or longer.

Various coating techniques may be applied to granules, beads, powders orpellets, tablets, capsules or combinations thereof containing activeagents to produce different and distinct release profiles. In someembodiments, the pharmaceutical composition is in a tablet or capsuleform containing a single coating layer. In other embodiments, thepharmaceutical composition is in a tablet or capsule form containingmultiple coating layers. In some embodiments, the pharmaceuticalcomposition of the present application is formulated forextended-release or delayed extended-release of up to 100% of the activeingredient.

In other embodiments, the pharmaceutical composition of the presentapplication is formulated for a two-phase extended-release or delayedtwo-phase extended-release characterized by an “immediate-release”component that is released within two hours of administration and an“extended-release” component which is released over a period of 2-12hours. In some embodiments, the “immediate-release” component providesabout 1-90% of the total dosage of the active agent(s) and the“extended-release” component provides 10-99% of the total dosage of theactive agent(s) to be delivered by the pharmaceutical formulation. Forexample, the immediate-release component may provide about 10-90%, orabout 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85% or 90% of the total dosage of the active agent(s) to bedelivered by the pharmaceutical formulation. The extended-releasecomponent provides about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the total dosageof the active agent(s) to be delivered by the formulation. In someembodiments, the immediate-release component and the extended-releasecomponent contain the same active ingredient. In other embodiments, theimmediate-release component and the extended-release component containdifferent active ingredients (e.g., one PG pathway inhibitor in onecomponent and another PG pathway inhibitor in another component). Insome embodiments, the immediate-release component and theextended-release component each contains a PG pathway inhibitor and ananalgesic selected from the group consisting of aspirin, ibuprofen,naproxen sodium, indomethacin, nabumetone, and acetaminophen. In otherembodiments, the immediate-release component and/or the extended-releasecomponent further comprises one or more additional active agentsselected from the groups consisting of an antimuscarinic agent, anantidiuretic, a spasmolytic, an inhibitor of phosphodiesterase type (PDE5 inhibitor) and zolpidem.

In some embodiments, the pharmaceutical composition comprises aplurality of active ingredients selected from the group consisting of PGpathway inhibitors, analgesics, antimuscarinic agents, antidiuretics,spasmolytics, PDE 5 inhibitors and zolpidem. Examples of antimuscarinicagents include, but are not limited to, oxybutynin, solifenacin,darifenacin, fesoterodine, tolterodine, trospium, atropine, andtricyclic antidepressants. Examples of antidiuretics include, but arenot limited to, antidiuretic hormone (ADH), angiotensin II, aldosterone,vasopressin, vasopressin analogs (e.g., desmopressin argipressin,lypressin, felypressin, ornipressin, terlipressin); vasopressin receptoragonists, atrial natriuretic peptide (ANP) and C-type natriureticpeptide (CNP) receptor (i.e., NPR1, NPR2, and NPR3) antagonists (e.g.,HS-142-1, isatin, [Asu7,23′]b-ANP-(7-28)], anantin, a cyclic peptidefrom Streptomyces coerulescens, and 3G12 monoclonal antibody);somatostatin type 2 receptor antagonists (e.g., somatostatin),pharmaceutically-acceptable derivatives, and analogs, salts, hydrates,and solvates thereof. Examples of spasmolytics include, but are notlimited to, carisoprodol, benzodiazepines, baclofen, cyclobenzaprine,metaxalone, methocarbamol, clonidine, clonidine analog, and dantrolene.Examples of PDE 5 inhibitors include, but are not limited to, tadalafil,sildenafil and vardenafil.

In some embodiments, the pharmaceutical composition comprises aplurality of active ingredients comprising (1) one or more PG pathwayinhibitors and (2) one or more other active ingredients selected fromthe group consisting of analgesics, antimuscarinic agents,antidiuretics, spasmolytics, PDE 5 inhibitors and zolpidem. In someembodiments, the plurality of active ingredients are formulated forimmediate-release. In other embodiments, the plurality of activeingredients are formulated for extended-release. In other embodiments,the plurality of active ingredients are formulated for delayed-release.In other embodiments, the plurality of active ingredients are formulatedfor both immediate-release and extended-release (e.g., a first portionof each active ingredient is formulated for immediate-release and asecond portion of each active ingredient is formulated forextended-release). In yet other embodiments, some of the plurality ofactive ingredients are formulated for immediate-release and some of theplurality of active ingredients are formulated for extended-release(e.g., active ingredients A, B, C are formulated for immediate-releaseand active ingredients C and D are formulated for extended-release). Insome other embodiments, the plurality of active ingredients areformulated for delayed-extended-release.

In certain embodiments, the pharmaceutical composition comprises animmediate-release component and an extended-release component. Theimmediate-release component may comprise one or more active ingredientsselected from the group consisting of PG pathway inhibitors, analgesics,antimuscarinic agents, antidiuretics, spasmolytics, PDE 5 inhibitors andzolpidem. The extended-release component may comprise one or more activeingredients selected from the group consisting of PG pathway inhibitors,analgesics, antimuscarinic agents, antidiuretics, spasmolytics, PDE 5inhibitors and zolpidem. In some embodiments, the immediate-releasecomponent and the extended-release component have exactly the sameactive ingredients. In other embodiments, the immediate-releasecomponent and the extended-release component have different activeingredients. In yet other embodiments, the immediate-release componentand the extended-release component have one or more common activeingredients. In some other embodiments, the immediate-release componentand/or the extended-release component is further coated with adelayed-release coating, such as an enteric coating. In otherembodiments, the pharmaceutical composition comprises two or more activeingredients formulated as two extended-release components, eachproviding a different extended-release profile. For example, a firstextended-release component releases a first active ingredient at a firstrelease rate and a second extended-release component releases a secondactive ingredient at a second release rate.

In some embodiments, the pharmaceutical composition comprises animmediate-release component and a delayed-release component. In otherembodiments, the pharmaceutical composition comprises two or more activeingredients formulated as two delayed-release components, each providinga different delayed-release profile. For example, a firstdelayed-release component releases a first active ingredient at a firsttime point, and a second delayed-release component releases a secondactive ingredient at a second time point.

The components in a combined release profile formulation (e.g.,formulations with a combination of an immediate-release component and anextended-release component, a combination of an immediate-releasecomponent and a delayed-release component, a combination of animmediate-release component, a delayed-release component, and anextended-release component, a combination of two or more delayed-releasecomponents, or a combination of two or more extended-release components)may contain the same active ingredient(s) or different activeingredient(s). In some embodiments, the immediate-release component mayprovide about 1% to about 80% of the total dosage of the active agent(s)to be delivered by the pharmaceutical formulation. In some embodiments,the combined release profile formulation contains an immediate-releasecomponent and the immediate-release component provide about 1% to about90%, about 10% to about 90%, about 20% to about 90%, about 30% to about90%, about 40% to about 90%, about 50% to about 90%, about 60% to about90%, about 70% to about 90% or about 80% to about 90% of the totaldosage of each active ingredient to be delivered by the formulation. Inalternate embodiments, the immediate-release component provides up toabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total dosageof each ingredient to be delivered by the formulation.

In some embodiments, the pharmaceutical formulation comprises an activecore comprised of one or more inert particles, each in the form of abead, pellet, pill, granular particle, microcapsule, microsphere,microgranule, nanocapsule, or nanosphere coated on its surfaces withdrugs in the form of e.g., a drug-containing film-forming compositionusing, for example, fluid bed techniques or other methodologies known tothose of skill in the art. The inert particle can be of various sizes,so long as it is large enough to remain poorly dissolved. Alternatively,the active core may be prepared by granulating and milling and/or byextrusion and spheronization of a polymer composition containing thedrug substance.

The amount of drug in the core will depend on the dose that is requiredand typically varies from about 5 to 90 weight %. Generally, thepolymeric coating on the active core will be from about 1 to 50% basedon the weight of the coated particle, depending on the lag time and typeof release profile required and/or the polymers and coating solventschosen. Those skilled in the art will be able to select an appropriateamount of drug for coating onto or incorporating into the core toachieve the desired dosage. In one embodiment, the inactive core may bea sugar sphere or a buffer crystal or an encapsulated buffer crystalsuch as calcium carbonate, sodium bicarbonate, fumaric acid, tartaricacid, which alters the microenvironment of the drug to facilitate itsrelease.

In some embodiments, the pharmaceutical composition comprises adelayed-release component formed by coating a water soluble/dispersibledrug-containing particle, such as a bead, with a mixture of a waterinsoluble polymer and an enteric polymer, wherein the water insolublepolymer and the enteric polymer may be present at a weight ratio of 4:1to 1:1, and the total weight of the coatings is 10 to 60 weight % basedon the total weight of the coated beads. The drug layered beads mayoptionally include an inner dissolution rate controlling membrane ofethylcellulose. The composition of the outer layer, as well as theindividual weights of the inner and outer layers of the polymericmembrane are optimized for achieving desired circadian rhythm releaseprofiles for a given active, which are predicted based on in vitro/invivo correlations.

In other embodiments, the formulations comprise a mixture ofimmediate-release drug-containing particles without a dissolution ratecontrolling polymer membrane and delayed release beads exhibiting, forexample, a lag time of 2-4 hours following oral administration, thusproviding a two-pulse release profile. In yet other embodiments, theformulations comprise a mixture of two types of delayed-release beads: afirst type that exhibits a lag time of 1-3 hours and a second type thatexhibits a lag time of 4-6 hours. In yet other embodiments, theformulations comprise a mixture of two types of release beads: a firsttype that exhibits immediate-release and a second type that exhibits alag time of 1-4 hours followed with extended-release.

In some embodiments, the formulations are designed with a releaseprofile such that a fraction of the active ingredient(s) (e.g., 10-80%)is released immediately or within two hours of administration, and therest is released over an extended period of time (e.g., over a period of2-24 hours). In other embodiments, the formulations are designed with arelease profile such that one active ingredient (e.g., an analgesic) arereleased immediately or within two hours of administration, and one ormore other active ingredients (e.g., a PG pathway inhibitor) arereleased over an extended period of time (e.g., over a period of 2-24hours).

The pharmaceutical composition may be administered daily or administeredon an as needed basis. The pharmaceutical composition may beadministered orally, intravenously, or intramuscularly. In preferredembodiments, the pharmaceutical composition is administered orally. Inother embodiments, the pharmaceutical composition is administered byretrograde perfusion through the urinary tract. In other embodiments,the pharmaceutical composition is administered by direct injection intobladder muscle.

In some embodiments, the pharmaceutical composition is administereddaily, twice a day or three times a day. In other embodiments, thepharmaceutical composition is administered every other day, every 3days, every 4 days, every 4 days, every 5 days, every 6 days, everyweek, every 2 weeks, every 3 weeks, every month, every 2 months or every3 months.

In some embodiments, the pharmaceutical composition is administered atbedtime. In some embodiments, the pharmaceutical composition isadministered within about two hours before bedtime, preferably withinabout one hour before bedtime. In another embodiment, the pharmaceuticalcomposition is administered about 2-4 hours before bedtime. In a furtherembodiment, the pharmaceutical composition is administered at least 4hours before bedtime.

The appropriate dosage (“therapeutically effective amount”) of theactive ingredient(s) in the immediate-release component, theextended-release component, the delayed-release component ordelayed-extended-release component will depend, for example, on theseverity and course of the condition, the mode of administration, thebioavailability of the particular ingredient(s), the age and weight ofthe patient, the patient's clinical history and response to the activeagent(s), discretion of the physician, etc.

As a general proposition, the therapeutically effective amount of the PGpathway inhibitor(s) in the immediate-release component, thedelayed-release component, the extended-release component or thedelayed-extended-release component is administered in the range of about1 μg/kg body weight/day to about 100 mg/kg body weight/day whether byone or more administrations. In some embodiments, the range of eachactive agent administered daily in a single dose or in multiple does isfrom about 1 μg/kg body weight/day to about 100 mg/kg body weight/day, 1μg/kg body weight/day to about 30 mg/kg body weight/day, 1 μg/kg bodyweight/day to about 10 mg/kg body weight/day, 1 μg/kg body weight/day toabout 3 mg/kg body weight/day, 1 μg/kg body weight/day to about 1 mg/kgbody weight/day, 1 μg/kg body weight/day to about 300 μg/kg bodyweight/day, 1 μg/kg body weight/day to about 100 μg/kg body weight/day,1 μg/kg body weight/day to about 30 μg/kg body weight/day, 1 μg/kg bodyweight/day to about 10 μg/kg body weight/day, 1 μg/kg body weight/day toabout 3 μg/kg body weight/day, 10 μg/kg body weight/day to about 100mg/kg body weight/day, 10 μg/kg body weight/day to about 30 mg/kg bodyweight/day, 10 μg/kg body weight/day to about 10 mg/kg body weight/day,10 μg/kg body weight/day to about 3 mg/kg body weight/day, 10 μg/kg bodyweight/day to about 1 mg/kg body weight/day, 10 μg/kg body weight/day toabout 300 μg/kg body weight/day, 10 μg/kg body weight/day to about 100μg/kg body weight/day, 10 μg/kg body weight/day to about 30 μg/kg bodyweight/day, 30 μg/kg body weight/day to about 100 mg/kg body weight/day,30 μg/kg body weight/day to about 30 mg/kg body weight/day, 30 μg/kgbody weight/day to about 10 mg/kg body weight/day, 30 μg/kg bodyweight/day to about 3 mg/kg body weight/day, 30 μg/kg body weight/day toabout 1 mg/kg body weight/day, 30 μg/kg body weight/day to about 300μg/kg body weight/day, 30 μg/kg body weight/day to about 100 μg/kg bodyweight/day, 100 μg/kg body weight/day to about 100 mg/kg bodyweight/day, 100 μg/kg body weight/day to about 30 mg/kg body weight/day,100 μg/kg body weight/day to about 10 mg/kg body weight/day, 100 μg/kgbody weight/day to about 3 mg/kg body weight/day, 100 μg/kg bodyweight/day to about 1 mg/kg body weight/day, 100 μg/kg body weight/dayto about 300 μg/kg body weight/day, 300 μg/kg body weight/day to about100 mg/kg body weight/day, 300 μg/kg body weight/day to about 30 mg/kgbody weight/day, 300 μg/kg body weight/day to about 10 mg/kg bodyweight/day, 300 μg/kg body weight/day to about 3 mg/kg body weight/day,300 μg/kg body weight/day to about 1 mg/kg body weight/day, 1 mg/kg bodyweight/day to about 100 mg/kg body weight/day, 1 mg/kg body weight/dayto about 30 mg/kg body weight/day, 1 mg/kg body weight/day to about 10mg/kg body weight/day, 1 mg/kg body weight/day to about 3 mg/kg bodyweight/day, 3 mg/kg body weight/day to about 100 mg/kg body weight/day,3 mg/kg body weight/day to about 30 mg/kg body weight/day, 3 mg/kg bodyweight/day to about 10 mg/kg body weight/day, 10 mg/kg body weight/dayto about 100 mg/kg body weight/day, 10 mg/kg body weight/day to about 30mg/kg body weight/day or 30 mg/kg body weight/day to about 100 mg/kgbody weight/day.

As a general proposition, the therapeutically effective amount of theanalgesic agent(s) in the immediate-release component, thedelayed-release component, the extended-release component or thedelayed-extended-release component is administered in the range of about10 μg/kg body weight/day to about 100 mg/kg body weight/day whether byone or more administrations. In some embodiments, the range of eachactive agent administered daily in a single dose or in multiple does isfrom about 10 μg/kg body weight/day to about 100 mg/kg body weight/day,10 μg/kg body weight/day to about 30 mg/kg body weight/day, 10 μg/kgbody weight/day to about 10 mg/kg body weight/day, 10 μg/kg bodyweight/day to about 3 mg/kg body weight/day, 10 μg/kg body weight/day toabout 1 mg/kg body weight/day, 10 μg/kg body weight/day to about 300μg/kg body weight/day, 10 μg/kg body weight/day to about 100 μg/kg bodyweight/day, 10 μg/kg body weight/day to about 30 μg/kg body weight/day,30 μg/kg body weight/day to about 100 mg/kg body weight/day, 30 μg/kgbody weight/day to about 30 mg/kg body weight/day, 30 μg/kg bodyweight/day to about 10 mg/kg body weight/day, 30 μg/kg body weight/dayto about 3 mg/kg body weight/day, 30 μg/kg body weight/day to about 1mg/kg body weight/day, 30 μg/kg body weight/day to about 300 μg/kg bodyweight/day, 30 μg/kg body weight/day to about 100 μg/kg body weight/day,100 μg/kg body weight/day to about 100 mg/kg body weight/day, 100 μg/kgbody weight/day to about 30 mg/kg body weight/day, 100 μg/kg bodyweight/day to about 10 mg/kg body weight/day, 100 μg/kg body weight/dayto about 3 mg/kg body weight/day, 100 μg/kg body weight/day to about 1mg/kg body weight/day, 100 μg/kg body weight/day to about 300 μg/kg bodyweight/day, 300 μg/kg body weight/day to about 100 mg/kg bodyweight/day, 300 μg/kg body weight/day to about 30 mg/kg body weight/day,300 μg/kg body weight/day to about 10 mg/kg body weight/day, 300 μg/kgbody weight/day to about 3 mg/kg body weight/day, 300 μg/kg bodyweight/day to about 1 mg/kg body weight/day, 1 mg/kg body weight/day toabout 100 mg/kg body weight/day, 1 mg/kg body weight/day to about 30mg/kg body weight/day, 1 mg/kg body weight/day to about 10 mg/kg bodyweight/day, 1 mg/kg body weight/day to about 3 mg/kg body weight/day, 3mg/kg body weight/day to about 100 mg/kg body weight/day, 3 mg/kg bodyweight/day to about 30 mg/kg body weight/day, 3 mg/kg body weight/day toabout 10 mg/kg body weight/day, 10 mg/kg body weight/day to about 100mg/kg body weight/day, 10 mg/kg body weight/day to about 30 mg/kg bodyweight/day or 30 mg/kg body weight/day to about 100 mg/kg bodyweight/day.

The analgesic agent(s) described herein may be included in animmediate-release component or an extended-release component, adelayed-release component, a delayed-extended-release component orcombinations thereof for daily oral administration at a single dose orcombined dose range of 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to 300 mg,1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3 mg, 3 mg to 2000mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mgto 10 mg, 10 mg to 2000 mg, 10 mg to 1000 mg, 10 mg to 300 mg, 10 mg to100 mg, 10 mg to 30 mg, 30 mg to 2000 mg, 30 mg to 1000 mg, 30 mg to 300mg, 30 mg to 100 mg, 100 mg to 2000 mg, 100 mg to 1000 mg, 100 mg to 300mg, 300 mg to 2000 mg, 300 mg to 1000 mg or 1000 mg to 2000 mg. Asexpected, the dosage will be dependent on the condition, size, age, andcondition of the patient.

In some embodiments, the pharmaceutical composition comprises a singleanalgesic agent. In one embodiment, the single analgesic agent isaspirin. In another embodiment, the single analgesic agent is ibuprofen.In another embodiment, the single analgesic agent is naproxen ornaproxen sodium. In another embodiment, the single analgesic agent isindomethacin. In another embodiment, the single analgesic agent isnabumetone. In another embodiment, the single analgesic agent isacetaminophen.

In other embodiments, the pharmaceutical composition comprises a pair ofanalgesic agents. Examples of such paired analgesic agents include, butare not limited to, acetylsalicylic acid and ibuprofen, acetylsalicylicacid and naproxen sodium, acetylsalicylic acid and nabumetone,acetylsalicylic acid and acetaminophen, acetylsalicylic acid andindomethancin, ibuprofen and naproxen sodium, ibuprofen and nabumetone,ibuprofen and acetaminophen, ibuprofen and indomethancin, naproxen,naproxen sodium and nabumetone, naproxen sodium and acetaminophen,naproxen sodium and indomethancin, nabumetone and acetaminophen,nabumetone and indomethancin, and acetaminophen and indomethancin. Thepaired analgesic agents are mixed at a weight ratio in the range of0.1:1 to 10:1, 0.2:1 to 5:1 or 0.3:1 to 3:1. In one embodiment, thepaired analgesic agents are mixed at a weight ratio of 1:1.

In some other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more antimuscarinic agents.Examples of the antimuscarinic agents include, but are not limited to,oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine,trospium, atropine, and tricyclic antidepressants. The daily dose ofantimuscarinic agent is in the range of 1 μg to 300 mg, 1 μg to 100 mg,1 μg to 30 mg; 1 μg to 10 mg, 1 μg to 3 mg, 1 μg to 1 mg, 1 μg to 300μg, 1 μg to 100 μg, 1 μg to 30 μg, 1 μg to 10 μg, 1 μg to 3 μg, 3 μg to100 mg, 3 μg to 100 mg, 3 μg to 30 mg; 3 μg to 10 mg, 3 μg to 3 mg, 3 μgto 1 mg, 3 μg to 300 μg, 3 μg to 100 μg, 3 μg to 30 μg, 3 μg to 10 μg,10 μg to 300 mg, 10 μg to 100 mg, 10 μg to 30 mg; 10 μg to 10 mg, 10 μgto 3 mg, 10 μg to 1 mg, 10 μg to 300 μg, 10 μg to 100 μg, 10 μg to 30μg, 30 μg to 300 mg, 30 μg to 100 mg, 30 μg to 30 mg; 30 μg to 10 mg, 30μg to 3 mg, 30 μg to 1 mg, 30 μg to 300 μg, 30 μg to 100 μg, 100 μg to300 mg, 100 μg to 100 mg, 100 μg to 30 mg; 100 μg to 10 mg, 100 μg to 3mg, 100 μg to 1 mg, 100 μg to 300 μg, 300 μg to 300 mg, 300 μg to 100mg, 300 μg to 30 mg; 300 μg to 10 mg, 300 μg to 3 mg, 300 μg to 1 mg, 1mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 3 mg, 3 mg to 300mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 300 mg, 10 mgto 100 mg, 10 mg to 30 mg, 30 mg to 300 mg, 30 mg to 100 mg or 100 mg to300 mg.

In some other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more antidiuretics. Examples of theantidiuretics include, but are not limited to, antidiuretic hormone(ADH), angiotensin II, aldosterone, vasopressin, vasopressin analogs(e.g., desmopressin argipressin, lypressin, felypressin, ornipressin,and terlipressin), vasopressin receptor agonists, atrial natriureticpeptide (ANP) and C-type natriuretic peptide (CNP) receptor (i.e., NPR1,NPR2, and NPR3) antagonists (e.g., HS-142-1, isatin,[Asu7,23′]b-ANP-(7-28)], anantin, a cyclic peptide from Streptomycescoerulescens, and 3G12 monoclonal antibody), somatostatin type 2receptor antagonists (e.g., somatostatin), pharmaceutically-acceptablederivatives, and analogs, salts, hydrates, and solvates thereof In someembodiments, the one or more antidiuretics comprise desmopressin. Inother embodiments, the one or more antidiuretics is desmopressin. Thedaily dose of antidiuretic is in the range of 1 μg to 300 mg, 1 μg to100 mg, 1 μg to 30 mg; 1 μg to 10 mg, 1 μg to 3 mg, 1 μg to 1 mg, 1 μgto 300 μg, 1 μg to 100 μg, 1 μg to 30 μg, 1 μg to 10 μg, 1 μg to 3 μg, 3μg to 100 mg, 3 μg to 100 mg, 3 μg to 30 mg; 3 μg to 10 mg, 3 μg to 3mg, 3 μg to 1 mg, 3 μg to 300 μg, 3 μg to 100 μg, 3 μg to 30 μg, 3 μg to10 μg, 10 μg to 300 mg, 10 μg to 100 mg, 10 μg to 30 mg; 10 μg to 10 mg,10 μg to 3 mg, 10 μg to 1 mg, 10 μg to 300 μg, 10 μg to 100 μg, 10 μg to30 μg, 30 μg to 300 mg, 30 μg to 100 mg, 30 μg to 30 mg; 30 μg to 10 mg,30 μg to 3 mg, 30 μg to 1 mg, 30 μg to 300 μg, 30 μg to 100 μg, 100 μgto 300 mg, 100 μg to 100 mg, 100 μg to 30 mg; 100 μg to 10 mg, 100 μg to3 mg, 100 μg to 1 mg, 100 μg to 300 μg, 300 μg to 300 mg, 300 μg to 100mg, 300 μg to 30 mg; 300 μg to 10 mg, 300 μg to 3 mg, 300 μg to 1 mg, 1mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 3 mg, 3 mg to 300mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 300 mg, 10 mgto 100 mg, 10 mg to 30 mg, 30 mg to 300 mg, 30 mg to 100 mg or 100 mg to300 mg.

In other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more spasmolytics. Examples ofspasmolytics include, but are not limited to, carisoprodol,benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol,clonidine, clonidine analog, and dantrolene. In some embodiments, thespasmolytics is used at a daily dose of 0.1 mg to 1000 mg, 0.1 mg to 300mg, 0.1 mg to 100 mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg to 3 mg,0.1 mg to 1 mg, 0.1 mg to 0.3 mg, 0.3 mg to 1000 mg, 0.3 mg to 300 mg,0.3 mg to 100 mg, 0.3 mg to 30 mg, 0.3 mg to 10 mg, 0.3 mg to 3 mg, 0.3mg to 1 mg, 1 mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to 30mg, 1 mg to 10 mg, 1 mg to 3 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mgto 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 1000 mg, 10 mg to 300mg, 10 mg to 100 mg, 10 mg to 30 mg, 30 mg to 1000 mg, 30 mg to 300 mg,30 mg to 100 mg, 100 mg to 1000 mg, 100 mg to 300 mg, or 300 mg to 1000mg.

In other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more PDE 5 inhibitors. Examples ofPDE 5 inhibitors include, but are not limited to, tadalafil, sildenafiland vardenafil. In some embodiments, the one or more PDE 5 inhibitorscomprise tadalafil. In other embodiments, the one or more PDE 5inhibitors is tadalafil. In some embodiments, the PDE 5 inhibitor isused at a daily dose of 0.1 mg to 1000 mg, 0.1 mg to 300 mg, 0.1 mg to100 mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg to 3 mg, 0.1 mg to 1mg, 0.1 mg to 0.3 mg, 0.3 mg to 1000 mg, 0.3 mg to 300 mg, 0.3 mg to 100mg, 0.3 mg to 30 mg, 0.3 mg to 10 mg, 0.3 mg to 3 mg, 0.3 mg to 1 mg, 1mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10mg, 1 mg to 3 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mgto 30 mg, 3 mg to 10 mg, 10 mg to 1000 mg, 10 mg to 300 mg, 10 mg to 100mg, 10 mg to 30 mg, 30 mg to 1000 mg, 30 mg to 300 mg, 30 mg to 100 mg,100 mg to 1000 mg, 100 mg to 300 mg, or 300 mg to 1000 mg.

In some other embodiments, the pharmaceutical composition of the presentapplication further comprises zolpidem. The daily dose of zolpidem is inthe range of 100 μg to 100 mg, 100 μg to 30 mg, 100 μg to 10 mg, 100 μgto 3 mg, 100 μg to 1 mg, 100 μg to 300 μg, 300 μg to 100 mg, 300 μg to30 mg, 300 μg to 10 mg, 300 μg to 3 mg, 300 μg to 1 mg, 1 mg to 100 mg,1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3 mg, 10 mg to 100 mg, 10 mg to 30mg, or 30 mg to 100 mg.

The antimuscarinic agents, antidiuretics, spasmolytics, zolpidem and/orPDE 5 inhibitors may be formulated, alone or together with other activeingredient(s) in the pharmaceutical composition, for immediate-release,extended-release, delayed release, delayed-extended-release orcombinations thereof

In certain embodiments, the pharmaceutical composition is formulated forextended release and comprises (1) an analgesic agent selected from thegroup consisting of cetylsalicylic acid, ibuprofen, naproxen, naproxensodium, nabumetone, acetaminophen, and indomethancin and (2) a PDE 5inhibitor, such as tadalafil.

The pharmaceutical composition may be formulated into a tablet, anorally disintegrating tablet, capsule, dragee, powder, granulate,liquid, gel or emulsion form. Said liquid, gel or emulsion may beingested by the subject in naked form or contained within a capsule.

In some embodiments, the pharmaceutical composition comprises a singleanalgesic agent and a single PDE 5 inhibitor. In one embodiment, thesingle analgesic agent is aspirin. In another embodiment, the singleanalgesic agent is ibuprofen. In another embodiment, the singleanalgesic agent is naproxen or naproxen sodium. In another embodiment,the single analgesic agent is indomethacin. In another embodiment, thesingle analgesic agent is nabumetone. In another embodiment, the singleanalgesic agent is acetaminophen. In another embodiment, the single PDE5 inhibitor is tadalafil. The analgesic agent and PDE 5 inhibitor may begiven at doses in the ranges described above.

In some embodiments, the pharmaceutical composition comprises one ormore analgesic agents, individually or in combination, in an amountbetween 10-1000 mg, 10-800 mg, 10-600 mg, 10-500 mg, 10-400 mg, 10-300mg, 10-250 mg, 10-200 mg, 10-150 mg, 10-100 mg 30-1000 mg, 30-800 mg,30-600 mg, 30-500 mg, 30-400 mg, 30-300 mg, 30-250 mg, 30-200 mg, 30-150mg, 30-100 mg, 100-1000 mg, 100-800 mg, 100-600 mg, 100-400 mg, 100-250mg, 300-1000 mg, 300-800 mg, 300-600 mg, 300-400 mg, 400-1000 mg,400-800 mg, 400-600 mg, 600-1000 mg, 600-800 mg or 800-1000 mg, whereinthe composition is formulated for extended release with a releaseprofile in which the one or more analgesic agents are releasedcontinuously over a period of 2-12 hours or 5-8 hours.

In some embodiments, the composition is formulated for extended-releasewith a release profile in which at least 90% of the one or moreanalgesic agents are released continuously over a period of 2-12 hoursor 5-8 hours.

In some embodiments, the composition is formulated for extended releasewith a release profile in which the one or more analgesic agents arereleased continuously over a period of 5, 6, 7, 8, 10 or 12 hours. Insome embodiments, the pharmaceutical composition further comprises anantimuscarinic agent, an antidiuretic, a spasmolytic, zolpidem or a PDE5 inhibitor.

In other embodiments, the composition is formulated for extended-releasewith a release profile in which the analgesic agent is released at asteady rate over a period of 2-12 hours or 5-8 hours. In otherembodiments, the composition is formulated for extended release with arelease profile in which the analgesic agent is released at a steadyrate over a period of 5, 6, 7, 8, 10 or 12 hours. As used herein, “asteady rate over a period of time” is defined as a release profile inwhich the release rate at any point during a given period of time iswithin 30% -300% of the average release rate over that given period oftime. For example, if 80 mg of aspirin is released at a steady rate overa period of 8 hours, the average release rate is 10 mg/hr during thisperiod of time and the actual release rate at any time during thisperiod is within the range of 3 mg/hr to 30 mg/hr (i.e., within 30% -300% of the average release rate of 10 mg/hr during the 8 hour period).In some embodiments, the pharmaceutical composition further comprises anantimuscarinic agent, an antidiuretic a spasmolytic, zolpidem or a PDE 5inhibitor.

In some embodiments, the analgesic agent is selected from the groupconsisting of aspirin, ibuprofen, naproxen sodium, naproxen,indomethacin, nabumetone and acetaminophen. In one embodiment, theanalgesic agent is acetaminophen. The pharmaceutical composition isformulated to provide a steady release of small amount of the analgesicagent to maintain an effective drug concentration in the blood such thatthe overall amount of the drug in a single dosage is reduced compared tothe immediate release formulation.

In some other embodiments, the pharmaceutical composition comprises oneor more analgesic agent(s), individually or in combination, in an amountbetween 10-1000 mg, 10-800 mg, 10-600 mg, 10-500 mg, 10-400 mg, 10-300mg, 10-250 mg, 10-200 mg, 10-150 mg, 10-100 mg 30-1000 mg, 30-800 mg,30-600 mg, 30-500 mg, 30-400 mg, 30-300 mg, 30-250 mg, 30-200 mg, 30-150mg, 30-100 mg, 100-1000 mg, 100-800 mg, 100-600 mg, 100-400 mg, 100-250mg, 300-1000 mg, 300-800 mg, 300-600 mg, 300-400 mg, 400-1000 mg,400-800 mg, 400-600 mg, 600-1000 mg, 600-800 mg or 800-1000 mg, whereinthe analgesic agent(s) are formulated for extended release,characterized by a two-phase release profile in which 20-80% of theanalgesic agent(s) are released within 2 hours of administration and theremainder are released continuously, or at a steady rate, over a periodof 2-12 hours or 5-8 hours. In yet another embodiment, the analgesicagent(s) is formulated for extended-release with a two-phase releaseprofile in which 20, 30, 40, 50 or 60% of the analgesic agent(s) arereleased within 2 hours of administration and the remainder are releasedcontinuously, or at a steady rate, over a period of 2-12 hours or 5-8hours. In one embodiment, the analgesic agent(s) are selected from thegroup consisting of aspirin, ibuprofen, naproxen sodium, naproxen,indomethacin, nabumetone, and acetaminophen. In one embodiment, theanalgesic agent is acetaminophen. In another embodiment, the analgesicagent is acetaminophen. In some embodiments, the pharmaceuticalcomposition further comprises an antimuscarinic agent, an antidiuretic,a spasmolytic, zolpidem and/or a PDE 5 inhibitor. In some embodiments,the antimuscarinic agent, antidiuretic, spasmolytic, zolpidem and/or PDE5 inhibitor is/are formulated for immediate-release.

Another aspect of the present application relates to a method forreducing frequency of urination by administering to a subject in needthereof, two or more PG pathway inhibitors alternatively to prevent thedevelopment of drug resistance. In one embodiment, the method comprisesadministering a first PG pathway inhibitor for a first period of timeand then administering a second PG pathway inhibitors for a secondperiod of time. In another embodiment, the method further comprisesadministering a third PG pathway inhibitor for a third period of time.The first, second, and third PG pathway inhibitors are different fromeach other and may be formulated for immediate-release,extended-release, delayed-release or combinations thereof.

Another aspect of the present application relates to a method fortreating nocturia by administering to a person in need thereof adiuretic, followed with the pharmaceutical composition of the presentapplication. The diuretic is dosed and formulated to have a diureticeffect within 6 hours of administration and is administered at least 8or 7 hours prior to bedtime. The pharmaceutical composition of thepresent application is formulated for extended-release or delayed,extended-release, and is administered within 2 hours prior to bedtime.

Examples of diuretics include, but are not limited to, acidifying salts,such as CaCl₂ and NH₄Cl; arginine vasopressin receptor 2 antagonists,such as amphotericin B and lithium citrate; aquaretics, such asGoldenrod and Juniper; Na—H exchanger antagonists, such as dopamine;carbonic anhydrase inhibitors, such as acetazolamide and dorzolamide;loop diuretics, such as bumetanide, ethacrynic acid, furosemide andtorsemide; osmotic diuretics, such as glucose and mannitol;potassium-sparing diuretics, such as amiloride, spironolactone,triamterene, potassium canrenoate; thiazides, such asbendroflumethiazide and hydrochlorothiazide; and xanthines, such ascaffeine, theophylline and theobromine.

Another aspect of the present application relates to a method forreducing the frequency of urination, comprising administering to asubject in need thereof an effective amount of the pharmaceuticalcomposition of the present application and an effective amount ofbotulinum toxin.

In some embodiments, the botulinum toxin is administered by injectioninto a bladder muscle; and orally administering to the subject thepharmaceutical composition of the present application. In someembodiments, the injecting step comprises injection of 10-200 units ofbotulinum toxin at 5-20 sites in bladder muscle with an injection doseof 2-10 units per site. In one embodiment, the injecting step comprisesinjection of botulinum toxin at 5 sites in bladder muscle with aninjection dose of 2-10 units per site. In another embodiment, theinjecting step comprises injection of botulinum toxin at 10 sites inbladder muscle at an injection dose of 2-10 units per site. In anotherembodiment, the injecting step comprises injection of botulinum toxin at15 sites in bladder muscle at an injection dose of 2-10 units per site.In yet another embodiment, the injecting step comprises injection ofbotulinum toxin at 20 sites in bladder muscle at an injection dose of2-10 units per site. In some embodiments, the injecting step is repeatedevery 3, 4, 6, 8, 10 or 12 months.

In some embodiments, the pharmaceutical composition comprises one ormore analgesic agents selected from the group consisting of aspirin,ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone andacetaminophen in an amount of 5-2000 mg per agent, and one or morespasmolytics selected from the group consisting of carisoprodol,benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol,clonidine, clonidine analog, and dantrolene in a total amount of 50-500mg, wherein the pharmaceutical composition is formulated for extendedrelease with a two-phase release profile in which 20-60% of the activeingredients are released within 2 hours of administration, and theremainder are released continuously, or at a steady rate, in a period of5-24 hours, 5-8 hours, 8-16 hours or 16-24 hours.

Another aspect of the present application relates to a pharmaceuticalcomposition that comprises a first component having an immediate-releasesubcomponent and an extended-release subcomponent, wherein the firstcomponent is formulated to release the subcomponents immediately afteradministration; and a second component comprising an immediate-releasesubcomponent and an extended-release subcomponent, wherein the secondcomponent is formulated for a delayed-release of the subcomponents. Insome embodiments, at least one of the subcomponents in the firstcomponent or the second component comprises an active ingredientcomprising one or more analgesic agents, and at least one of thesubcomponents in the first component or the second component comprisesan active ingredient comprising a PG pathway inhibitor, such as a PGinhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments, thePG pathway inhibitor is selected from the groups consisting ofinhibitors of PG activity, inhibitors of PG synthesis, inhibitors of PGTactivity, inhibitors of PGT expression, inhibitors of PGR activity, andinhibitors of PGR expression.

In some embodiments, each of the subcomponents in the first component orthe second component comprises an active ingredient comprising one ormore analgesic agents and/or a PG pathway inhibitor, such as a PGinhibitor, a PGT inhibitor or a PGR inhibitor.

In some embodiments, the one or more analgesic agents are selected fromthe group consisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone, and acetaminophen.

In some related embodiments, the immediate-release subcomponent and theextended-release subcomponent in the first component each comprises anactive ingredient comprising one or more analgesic agents, and/or a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor. In other embodiments, the immediate-release subcomponent andthe extended-release subcomponent in the second component each comprisesan active ingredient comprising one or more analgesic agents, and/or aPG pathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor.

In some embodiments, the one or more analgesic agents compriseacetaminophen. In yet other embodiments, at least one of thesubcomponents in the first component or the second component comprisesan active ingredient comprising one or more analgesic agents and a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor.

In some related embodiments, the second component is coated with anenteric coating.

In some related embodiments, the second component is formulated torelease the subcomponents after a lag time of 1-4 or 2-4 hours or 4-8hours following oral administration.

In some related embodiments, the extended-release subcomponent in thefirst component is formulated to release its active ingredient over atime interval of about 2-10 hours.

In some related embodiments, the extended-release subcomponent in thesecond component is formulated to release its active ingredient over atime interval of about 2-10 hours.

In some related embodiments, the active ingredient in theimmediate-release subcomponent and the extended-release subcomponent inthe first component further comprises an antimuscarinic agent. In someembodiments, the active ingredient in the immediate-release subcomponentand the extended-release subcomponent in the second component furthercomprises an antimuscarinic agent. In some embodiments, the activeingredient in the immediate-release subcomponent and theextended-release subcomponent in both the first and the second componentfurther comprises an antimuscarinic agent.

In some related embodiments, the active ingredient in theimmediate-release subcomponent and the extended-release subcomponent inthe first component further comprises an antidiuretic agent. In someembodiments, the active ingredient in the immediate-release subcomponentand the extended-release subcomponent in the second component furthercomprises an antidiuretic agent. In some embodiments, the activeingredient in the immediate-release subcomponent and theextended-release subcomponent in both the first and the second componentfurther comprises an antidiuretic agent.

In some related embodiments, the active ingredient in theimmediate-release subcomponent and the extended-release subcomponent inthe first component further comprises a spasmolytic. In someembodiments, the active ingredient in the immediate-release subcomponentand the extended-release subcomponent in the second component furthercomprises a spasmolytic. In some embodiments, the active ingredient inthe immediate-release subcomponent and the extended-release subcomponentin both the first and the second component further comprises aspasmolytic.

In some related embodiments, the immediate-release subcomponent and theextended-release subcomponent in the first component each comprises ananalgesic agent, such as acetaminophen, in an amount of 5-2000 mg. Insome embodiments, the immediate-release subcomponent and theextended-release subcomponent in the second component each comprises ananalgesic agent, such as acetaminophen, in an amount of 5-2000 mg. Insome embodiments, the active ingredient in the immediate-releasesubcomponent and the extended-release subcomponent in both the first andthe second component each comprises an analgesic agent, such asacetaminophen, in an amount of 5-2000 mg.

In some related embodiments, the active ingredient in theimmediate-release subcomponent of the first component and the activeingredient in the immediate-release subcomponent of the second componentboth comprise an analgesic agent, such as acetaminophen. In someembodiments, the active ingredient in the immediate-release subcomponentof the first component and the active ingredient in theimmediate-release subcomponent of the second component comprisedifferent analgesic agents.

Another aspect of the present application relates to a pharmaceuticalcomposition that comprises a first component comprising animmediate-release subcomponent, wherein the immediate-releasesubcomponent comprises an active ingredient comprising one or moreagents selected from the group consisting of analgesic agents and a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor, wherein the first component is formulated to release itssubcomponent immediately after oral administration; and a secondcomponent comprising an immediate-release subcomponent and anextended-release subcomponent, wherein the second component isformulated to release its subcomponent after gastric emptying, whereinthe subcomponents in the second component each comprises an activeingredient comprising one or more agents selected from the groupconsisting of analgesic agents and a PG pathway inhibitor, such as a PGinhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments, thePG pathway inhibitor is selected from the groups consisting ofinhibitors of PG activity, inhibitors of PG synthesis, inhibitors of PGTactivity, inhibitors of PGT expression, inhibitors of PGR activity, andinhibitors of PGR expression.

In some embodiments, the one or more analgesic agents are selected fromthe group consisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone, and acetaminophen.

In some related embodiments, the second component is formulated torelease the subcomponents after a lag time of 2-12 hours, 2-4 hours, 2-6hours, 2-8 hours, or 4-8 hours following oral administration.

In some related embodiments, the active ingredient in theimmediate-release subcomponent and the extended-release subcomponent ofthe second component comprises one or more analgesic agents.

In some related embodiments, the first component further comprises anextended-release subcomponent, wherein the extended-release subcomponentcomprises an active ingredient comprising one or more agents selectedfrom the group consisting of analgesic agents and a PG pathwayinhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor.In some embodiments, the one or more agents comprises an analgesic agentselected from the group consisting of aspirin, ibuprofen, naproxen,naproxen sodium, indomethacin, nabumetone, and acetaminophen.

In some embodiments, the immediate-release subcomponent and theextended-release subcomponent in the second component each comprises aPG pathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor.

In some related embodiments, at least one of the active ingredients inthe immediate-release subcomponent and/or the extended-releasesubcomponent of the first and the second components further comprises anagent selected from the group consisting of antimuscarinic agents,antidiuretic agents and spasmolytics.

In some related embodiments, the active ingredient in theimmediate-release subcomponent and/or the extended-release subcomponentof the first component further comprises an agent selected from thegroup consisting of antimuscarinic agents, antidiuretic agents andspasmolytics.

In some related embodiments, the active ingredient in theimmediate-release subcomponent and/or the extended-release subcomponentof the second component further comprises an agent selected from thegroup consisting of antimuscarinic agents, antidiuretic agents andspasmolytics.

Another aspect of the present application relates to a pharmaceuticalcomposition that comprises a first component comprising animmediate-release subcomponent and an extended-release subcomponent,wherein the first component is formulated to release the subcomponentsimmediately after administration; and a second component comprising animmediate-release subcomponent and an extended-release subcomponent,wherein the second component is formulated for a delayed-release of thesubcomponents, wherein the immediate-release subcomponent and theextended-release subcomponent in the first component each comprises anactive ingredient comprising one or more analgesic agents and a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor, and wherein the immediate-release subcomponent and theextended-release subcomponent in the second component each comprises anactive ingredient comprising one or more analgesic agents and a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor, wherein the pharmaceutical composition reduces the frequencyof urination in patients in need thereof. In some embodiments, the PGpathway inhibitor is selected from the groups consisting of inhibitorsof PG activity, inhibitors of PG synthesis, inhibitors of PGT activity,inhibitors of PGT expression, inhibitors of PGR activity, and inhibitorsof PGR expression.

In some embodiments, the one or more analgesic agents are selected fromthe group consisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone, and acetaminophen. In some embodiments, theone or more analgesic agents comprise acetaminophen.

In other embodiments, the pharmaceutical composition comprises a pair ofanalgesic agents. Examples of such paired analgesic agents include, butare not limited to, acetaminophen and an NSAID, acetylsalicylic acid andibuprofen, acetylsalicylic acid and naproxen sodium, acetylsalicylicacid and nabumetone, acetylsalicylic acid and acetaminophen,acetylsalicylic acid and indomethancin, ibuprofen and naproxen sodium,ibuprofen and nabumetone, ibuprofen and acetaminophen, ibuprofen andindomethancin, naproxen sodium and nabumetone, naproxen sodium andacetaminophen, naproxen sodium and indomethancin, nabumetone andacetaminophen, nabumetone and indomethancin, and acetaminophen andindomethancin. The paired analgesic agents are mixed at a weight ratioin the range of 0.1:1 to 10:1, 0.2:1 to 5:1 or 0.3:1 to 3:1 with acombined dose or single dose (i.e., the dose for each analgesic) in therange of 5 mg to 2000 mg, 20 mg to 2000 mg, 100 mg to 2000 mg, 200 mg to2000 mg, 500 mg to 2000 mg, 5 mg to 1500 mg, 20 mg to 1500 mg, 100 mg to1500 mg, 200 mg to 1500 mg, 500 mg to 1500 mg, 5 mg to 1000 mg, 20 mg to1000 mg, 100 mg to 1000 mg, 250 mg to 500 mg, 250 mg to 1000 mg, 250 mgto 1500 mg, 500 mg to 1000 mg, 500 mg to 1500 mg, 1000 mg to 1500 mg,and 1000 mg to 2000 mg. In one embodiment, the paired analgesic agentsare mixed at a weight ratio of 1:1.

Another aspect of the present application relates to a pharmaceuticalcomposition that comprises an immediate-release component and anextended-release component. Each component comprises a pair of analgesicagents as described above and a PG pathway inhibitor, such as a PGinhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments, theimmediate-release component and the extended-release component comprisedifferent pairs of analgesic agents. In some embodiments, theimmediate-release component and the extended-release component comprisethe same pair of analgesic agents. In some embodiments, theimmediate-release component and the extended-release component eachcomprises acetaminophen and an NSAID. In some embodiments, theimmediate-release component and the extended-release component eachcomprises acetaminophen and ibuprofen. In some embodiments, theimmediate-release component and the extended-release component eachconsists of acetaminophen, ibuprofen and a PG pathway inhibitor, such asa PG inhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments,the PG pathway inhibitor is selected from the groups consisting ofinhibitors of PG activity, inhibitors of PG synthesis, inhibitors of PGTactivity, inhibitors of PGT expression, inhibitors of PGR activity, andinhibitors of PGR expression.

In some embodiments, the extended-release component is formulated forextended release over a period of 0.5-24, 2-6, 6-10, 10-14, or 14-24hours. In some embodiments, the extended-release component is formulatedfor extended release over a period of about 8 hours. In someembodiments, the extended-release component is coated with adelayed-release coating. In some embodiments, the delayed-releasecoating delays the release of the extended-release component for aperiod of 0.1-12, 0.5-12, 1-12, 2-12, 1-4, 2-4, 4-8 or 8-12 hours. Insome embodiments, the delayed-release coating is an enteric coating. Insome embodiments, the pharmaceutical composition with animmediate-release component and an extended-release component isformulated into an orally disintegrating tablet.

As used herein, the term “orally disintegrating tablet” or “orallydisintegrating formulation” refers to drug tablet or formulation thatrapidly disintegrates or dissolves in the oral cavity. Orallydisintegrating formulations differ from traditional tablets in that theyare designed to be dissolved on the tongue rather than swallowed whole.In some embodiments, the orally disintegrating formulations are designedto completely disintegrate or dissolve in the oral cavity without theaid of additional water (i.e., in saliva only) in 5, 10, 20, 30, 60, 90,120, 180, 240 or 300 seconds.

In some embodiments, the pharmaceutical composition with animmediate-release component and an extended-release component isformulated into a liquid form for oral administration. Examples of theliquid form formulation include, but are not limited to, gels, emulsionsand particle suspensions. For example, the extended-release componentmay be formulated into a gel form that solidifies in the stomach. Insome embodiments, the pharmaceutical composition with animmediate-release component and an extended-release component isformulated into a pixie pack of powder that can quickly melt on thetongue. In some embodiments, the immediate-release component or theextended release component or both further comprise one or moreadditional agents selected from the group consisting of antimuscarinicagents, spasmolytics and antidiuretic agents.

Method of Manufacture

Another aspect of the present application relates to methods ofmanufacturing extended-release pharmaceutical compositions for reducingthe frequency of urination. In some embodiments, the method comprisesthe steps of forming a first mixture having a first active ingredientformulated for immediate release and a second active ingredientformulated for extended release; coating the first mixture with adelayed release coating to form a core structure; and then coating thecore structure with a second mixture comprising a third activeingredient formulated for immediate release and a fourth activeingredient formulated for extended release. In one embodiment, at leastone of the first, second, third and fourth active ingredients comprisesan analgesic agent and at least one of the first, second, third andfourth active ingredients comprises a PG pathway inhibitor, such as a PGinhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments, thePG pathway inhibitor is selected from the groups consisting ofinhibitors of PG activity, inhibitors of PG synthesis, inhibitors of PGTactivity, inhibitors of PGT expression, inhibitors of PGR activity, andinhibitors of PGR expression.

In some embodiments, the analgesic agent is selected from the groupconsisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone and acetaminophen, and wherein at least one ofthe first, second, third and fourth active ingredients comprises 5 mg to2000 mg of the analgesic agent.

In some embodiments, at least one of the first, second, third and fourthactive ingredients comprises (1) an analgesic agent selected from thegroup consisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone, and acetaminophen, and (2) a PG pathwayinhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor.

In some embodiments, the at least one of the first, second, third andfourth active ingredients comprises (1) acetaminophen, and (2) a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor.

In some embodiments, the at least one of the first, second, third andfourth active ingredients comprises an agent selected from the groupconsisting antimuscarinic agents, antidiuretic agents and spasmolytics.

In some embodiments, the delayed release coating is an enteric coating.In some embodiments, the enteric coating comprises a pH-dependentpolymer. In some embodiments, the delayed release coating comprises aswelling layer covered by an outer semi-permeable polymer layer. In someembodiments, the delayed release coating is formulated to release thecoated material after a lag time of 0.1-12 hours, 0.5-12 hours, 1-12hours, 2-12 hours, 1-4 hours, 2-4 hours, 2-6 hours, 2-8 hours, 4-6 hoursor 4-8 hours after oral administration.

In some embodiments, the second active ingredient, or the fourth activeingredient or both comprise an active core comprising anextended-release coating or a polymeric matrix effecting diffusioncontrolled release.

In some embodiments, the first mixture is prepared by mixing the firstactive ingredient in liquid or powder form with the second activeingredient, which is formulated for extended release. As describedabove, the second active ingredient may be formulated in an extendedrelease formulation having an active core comprised of one or more inertparticles, each in the form of a bead, pellet, pill, granular particle,microcapsule, microsphere, microgranule, nanocapsule, or nanospherecoated on its surfaces with drugs in the form of e.g., a drug-containingcoating or film-forming composition using, for example, fluid bedtechniques or other methodologies known to those of skill in the art.The inert particle can be of various sizes, so long as it is largeenough to remain undissolved. Alternatively, the active core may beprepared by granulating and milling and/or by extrusion andspheronization of a polymer composition containing the drug substance.In some embodiments, the active core comprises an extended-releasecoating or a polymeric matrix effecting diffusion controlled release, asdescribed in more detail earlier. In some embodiments, the polymericmatrix is a water soluble or water-swellable matrix. In someembodiments, the second active ingredient is simply mixed with the firstactive ingredient. Either ingredient or both ingredients may be in theform of bead, pellet, granular particle, pill, microcapsule,microsphere, microgranule, nanocapsule or nanosphere as a powder or as aliquid suspension. In other embodiments, the second active ingredientform an active core that is coated with the first active ingredient. Insome embodiments, the second active ingredient in the first mixture isformulated to release the active ingredient over a period of 2-4 hours,2-6 hours, 2-8 hours or 2-10 hours.

In some embodiments, the second active ingredient is kept in acompartment partially or completely separate from the first activeingredient. In other embodiments, the first mixture is formed by keepingthe second active ingredient in a compartment partially or completelyseparated from the first active ingredient.

The first mixture is then coated with a delayed release coating to forma core structure. In some embodiments, the delayed release coating is anenteric coating. In some embodiments, the enteric coating comprises apH-dependent polymer that maintains its structure integrity at low pH,such as the pH in the stomach (normally in the range of 1.5-3.5). Insome embodiments, the term “low pH” refers to a pH value of 4.0, 3.5,3.0, 2.5, 2.0, 1.5, 1.0 or lower. In some embodiments, the entericcoating comprises one or more pH-dependent polymers and one or morepolysaccharides that are resistant to erosion in both the stomach andintestine, thus allowing the release of the first mixture only in thecolon. In some embodiments, the delayed release coating comprises two ormore layers of coating. In some embodiments, the delayed release coatingcomprises a swelling layer and an outer semi-permeable polymer layerthat covers the swelling layer.

In the next step, the coated core structure is re-coated with a secondmixture that comprises a third active ingredient formulated forimmediate release and a fourth active ingredient formulated for extendedrelease. In some embodiments, the second mixture is prepared by mixingthe third active ingredient in liquid or powder form with the fourthactive ingredient, which is formulated for extended release. The fourthactive ingredient may be formulated in an extended release formulationhaving an active core comprised of one or more inert particles, each inthe form of a bead, pellet, pill, granular particle, microcapsule,microsphere, microgranule, nanocapsule, or nanosphere coated on itssurfaces with drugs in the form of e.g., a drug-containing coating orfilm-forming composition using, for example, fluid bed techniques orother methodologies known to those of skill in the art. The inertparticle can be of various sizes, so long as it is large enough toremain poorly dissolved. Alternatively, the active core may be preparedby granulating and milling and/or by extrusion and spheronization of apolymer composition containing the drug substance. In some embodiments,the active core comprises an extended-release coating or a polymericmatrix effecting diffusion controlled release, as described in moredetail earlier. In some embodiments, the polymeric matrix is a watersoluble or water-swellable matrix. In some embodiments, the fourthactive ingredient is simply mixed with the third active ingredient.Either ingredient or both ingredients may be in the form of bead,pellet, granular particle, pill, microcapsule, microsphere,microgranule, nanocapsule or nanosphere as a powder or as a liquidsuspension.

In other embodiments, the coated core structure is re-coated first withthe fourth active ingredient, and then coated with the third activeingredient. In some embodiments, the fourth active ingredient isformulated to release the active ingredient over a period of 2-4 hours,2-6 hours, 2-8 hours or 2-10 hours.

In some embodiments, the fourth active ingredient is kept in acompartment partially or completely separated from the third activeingredient. In other embodiments, the second mixture is formed bykeeping the fourth active ingredient in a compartment partially orcompletely separated from the third active ingredient.

In other embodiments, the method comprises the steps of forming a corestructure comprising a first active ingredient formulated for immediaterelease and a second active ingredient formulated for extended release,coating the core structure with a delayed release coating to form acoated core structure, and mixing the coated core structure with a thirdactive ingredient formulated for immediate release and a fourth activeingredient formulated for extended release. The first, second, third andfourth active ingredients can be the active ingredients described above.In one embodiment, the first, second, third and fourth activeingredients each comprises an analgesic agent and/or a PG pathwayinhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor.In some embodiments, the PG pathway inhibitor is selected from thegroups consisting of inhibitors of PG activity, inhibitors of PGsynthesis, inhibitors of PGT activity, inhibitors of PGT expression,inhibitors of PGR activity, and inhibitors of PGR expression. In someembodiments, the analgesic agent is selected from the group consistingof aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin,nabumetone and acetaminophen. In some embodiments, the method furthercomprises the step of preparing a dosage form with the final mixture. Insome embodiments, the dosage form is in a tablet form. In someembodiments, the dosage form is in an orally disintegrating form, e.g.,orally disintegrating tablet form. In some embodiments, the dosage formis in a beads-in-a-capsule form. In some embodiments, the dosage form isin a liquid (e.g., emulsion) form.

In other embodiments, the method comprises the steps of forming a corestructure comprising a first active ingredient formulated for immediaterelease and a second active ingredient formulated for extended release,coating the core structure with a delayed release coating to form acoated core structure, mixing the coated core structure with a thirdingredient formulated for immediate release and a fourth ingredientformulated for extended release.

Another aspect of the present application relates to a method formanufacturing a pharmaceutical composition for reducing the frequency ofurination. The method comprises the step of forming a core structurecomprising a first active ingredient formulated for immediate releaseand a second active ingredient formulated for extended release; coatingthe core structure with a delayed release coating to form a coated corestructure; mixing the coated core structure with a third activeingredient formulated for immediate release and a fourth activeingredient formulated for extended release to form a final mixture, andcompressing the final mixture into a tablet. In some embodiments, atleast one of the first, second, third and fourth active ingredientscomprises an analgesic agent and at least one of the first, second,third and fourth active ingredients comprises a PG pathway inhibitor,such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor. In someembodiments, the PG pathway inhibitor is selected from the groupsconsisting of inhibitors of PG activity, inhibitors of PG synthesis,inhibitors of PGT activity, inhibitors of PGT expression, inhibitors ofPGR activity, and inhibitors of PGR expression.

In some embodiments, the analgesic agent is selected from the groupconsisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone and acetaminophen and wherein at least one ofthe first, second, third and fourth active ingredients comprises 5-2000mg of the analgesic agent.

In some embodiments, the at least one of the first, second, third, andfourth active ingredients comprises: (1) acetaminophen; and (2) a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor.

In some embodiments, the at least one of the first, second, third andfourth active ingredients comprises an agent selected from the groupconsisting of antimuscarinic agents, antidiuretic agents andspasmolytics.

Another aspect of the present application relates to a method formanufacturing a pharmaceutical composition for reducing the frequency ofurination. The method comprises the steps of forming a core structurecomprising a first active ingredient formulated for immediate releaseand a second active ingredient formulated for extended release; coatingthe core structure with a delayed release coating to form a coated corestructure; coating the coated core structure with a third activeingredient formulated for immediate release to form a double-coated corestructure. In some embodiments, wherein at least one of the first,second and third active ingredients comprises an analgesic agent and atleast one of the first, second and third active ingredients comprises aPG pathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor. In some embodiments, the PG pathway inhibitor is selectedfrom the groups consisting of inhibitors of PG activity, inhibitors ofPG synthesis, inhibitors of PGT activity, inhibitors of PGT expression,inhibitors of PGR activity, and inhibitors of PGR expression.

In some embodiments, the analgesic agent is selected from the groupconsisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone and acetaminophen and wherein at least one ofthe first, second and third active ingredients comprises 5-2000 mg ofthe analgesic agent.

In some embodiments, at least one of the first, second and third activeingredients comprises: (1) acetaminophen; and (2) a PG pathwayinhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor.

In some embodiments, the at least one of the first, second and thirdactive ingredients comprises an agent selected from the group consistingantimuscarinic agents, antidiuretic agents and spasmolytics.

Another aspect of the present application relates to a method formanufacturing a pharmaceutical composition for reducing the frequency ofurination. The method comprises the steps of forming a core structurecomprising a first pair of analgesic agents formulated forextended-release, and coating the core structure with a coating layercomprising a second pair of analgesics, wherein the second pair ofanalgesics is formulated for immediate release and wherein either thecore structure or the coating layer or both further comprise a PGpathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor. In some embodiments, the PG pathway inhibitor is selectedfrom the groups consisting of inhibitors of PG activity, inhibitors ofPG synthesis, inhibitors of PGT activity, inhibitors of PGT expression,inhibitors of PGR activity, and inhibitors of PGR expression.

In some embodiments, the core structure is first coated with adelayed-release coating and then coated with a coating layer comprisinga second pair of analgesics, wherein the second pair of analgesics isformulated for immediate release.

In some embodiments, the method comprises the steps of forming a firstmixture comprising a first pair of analgesic agents formulated forextended-release, forming a second mixture comprising a second pair ofanalgesic agents formulated for immediate-release, and combining thefirst mixture and the second mixture to form a final mixture, whereineither the first mixture or the second mixture or both further comprisea PG pathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGRinhibitor.

In some embodiments, the first mixture, the second mixture and the finalmixture are mixtures of solid materials. In some embodiments, the finalmixture is in powder or granulate form. In some embodiments, the methodfurther comprises the step of pressing the final mixture into a tabletform. In some embodiments, the final mixture is in a liquid, gel oremulsion form.

Examples of paired analgesic agents include, but are not limited to,acetaminophen and an NSAID, acetylsalicylic acid and ibuprofen,acetylsalicylic acid and naproxen sodium, acetylsalicylic acid andnabumetone, acetylsalicylic acid and acetaminophen, acetylsalicylic acidand indomethancin, ibuprofen and naproxen sodium, ibuprofen andnabumetone, ibuprofen and acetaminophen, ibuprofen and indomethancin,naproxen sodium and nabumetone, naproxen sodium and acetaminophen,naproxen sodium and indomethancin, nabumetone and acetaminophen,nabumetone and indomethancin, and acetaminophen and indomethancin. Insome embodiments, the first pair of analgesic agents is different fromthe second pair of analgesic agents. In other embodiments, the firstpair of analgesic agents is the same as the second pair of analgesicagents. In one embodiment, the first pair of analgesic agents and thesecond pair of analgesic agents are both acetaminophen and ibuprofen.

For example, the extended-release component may be formulated into a gelform that solidifies in the stomach. In some embodiments, thepharmaceutical composition with an immediate-release component and anextended-release component is formulated into a pixie pack of powderthat can quickly melt on the tongue. In some embodiments, thepharmaceutical composition with an immediate-release component and anextended-release component is formulated into an orally disintegratingtablet using loose compression tableting. In loose compression, orallydisintegrating formulation is compressed at much lower forces (4-20 kN)than traditional tablets. In some embodiments, the orally disintegratingformulation contains some form of sugar, such as mannitol, to improvemouth feel. In some embodiments, the orally disintegrating tablet isproduced using lyophilized orally disintegrating formulation.

The present invention is further illustrated by the following examplewhich should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application are incorporated herein by reference.

EXAMPLE 1 Inhibition of the Urge to Urinate with Ibuprofen

Twenty volunteer subjects, both male and female were enrolled, each ofwhich experienced a premature urge or desire to urinate, interferingwith their ability to sleep for a sufficient period of time to feeladequately rested. Each subject ingested 400-800 mg of ibuprofen as asingle dose prior to bedtime. At least 14 subjects reported that theywere able to rest better because they were not being awakened asfrequently by the urge to urinate.

Several subjects reported that after several weeks of nightly use ofibuprofen, the benefit of less frequent urges to urinate was no longerbeing realized. However, all of these subjects further reported thereturn of the benefit after several days of abstaining from taking thedosages. More recent testing has confirmed similar results can beachieved at much lower dosages without any subsequent diminution ofbenefits.

EXAMPLE 2 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Macrophage Responses to Inflammatory AndNon-Inflammatory Stimuli

Experimental Design

This study is designed to determine the dose and in vitro efficacy ofanalgesics and antimuscarinic agents in controlling macrophage responseto inflammatory and non-inflammatory stimuli mediated by COX2 andprostaglandins (PGE, PGH, etc.). It establishes baseline (dose andkinetic) responses to inflammatory and non-inflammatory effectors inbladder cells. Briefly, cultured cells are exposed to analgesic agentsand/or antimuscarinic agents in the absence or presence of variouseffectors.

The effectors include: lipopolysaccharide (LPS), an inflammatory agent,and Cox2 inducer as inflammatory stimuli; carbachol or acetylcholine,stimulators of smooth muscle contraction as non-inflammatory stimuli;botulinum neurotoxin A, a known inhibitor of acetylcholine release, aspositive control; and arachidonic acid (AA), gamma linolenic acid(DGLA), or eicosapentaenoic acid (EPA) as precursors of prostaglandins,which are produced following the sequential oxidation of AA, DGLA, orEPA inside the cell by cyclooxygenases (COX1 and COX2) and terminalprostaglandin synthases.

The analgesic agents include: Salicylates such as aspirin;iso-butyl-propanoic-phenolic acid derivative (ibuprofen) such as Advil,Motrin, Nuprin, and Medipren; naproxen sodium such as Aleve, Anaprox,Antalgin, Feminax Ultra, Flanax, Inza, Midol Extended Relief, Nalgesin,Naposin, Naprelan, Naprogesic, Naprosyn, Naprosyn suspension,EC-Naprosyn, Narocin, Proxen, Synflex and Xenobid; acetic acidderivative such as indomethacin (Indocin);1-naphthaleneacetic acidderivative such as nabumetone or relafen; N-acetyl-para-aminophenol(APAP) derivative such as acetaminophen or paracetamol (Tylenol); andCelecoxib.

The antimuscarinic agents include oxybutynin, solifenacin, darifenacin,and atropine.

Macrophages are subjected to short term (1-2 hrs) or long term (24-48hrs) stimulation with:

-   -   (1) Each analgesic agent alone at various doses.    -   (2) Each analgesic agent at various doses in the presence of        LPS.    -   (3) Each analgesic agent at various doses in the presence of        carbachol or acetylcholine.    -   (4) Each analgesic agent at various doses in the presence of AA,        DGLA, or EPA.    -   (5) Botulinum neurotoxin A alone at various doses.    -   (6) Botulinum neurotoxin A at various doses in the presence of        LPS.    -   (7) Botulinum neurotoxin A at various doses in the presence of        carbachol or acetylcholine.    -   (8) Botulinum neurotoxin A at various doses in the presence of        AA, DGLA, or EPA.    -   (9) Each antimuscarinic agent alone at various doses.    -   (10) Each antimuscarinic agent at various doses in the presence        of LPS.    -   (11) Each antimuscarinic agent at various doses in the presence        of carbachol or acetylcholine.    -   (12) Each antimuscarinic agent at various doses in the presence        of AA, DGLA, or EPA.

The cells are then analyzed for the release of PGH₂; PGE; PGE₂;Prostacydin; Thromboxane; IL-1β; IL-6; TNF-α; the COX2 activity; theproduction of cAMP and cGMP; the production of IL-1β, IL-6, TNF-α, andCOX2 mRNA; and surface expression of CD80, CD86, and MHC class IImolecules.

Materials and Methods

Macrophage Cells

Murine RAW264.7 or J774 macrophage cells (obtained from ATCC) were usedin this study. Cells were maintained in a culture medium containing RPMI1640 supplemented with 10% fetal bovine serum (FBS), 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin. Cellswere cultured at 37° C. in a 5% CO₂ atmosphere and split (passages) oncea week.

In Vitro Treatment of Macrophage Cells with Analgesics

RAW264.7 macrophage cells were seeded in 96-well plates at a celldensity of 1.5×10⁵ cells per well in 100 μl of the culture medium. Thecells were treated with (1) various concentrations of analgesic(acetaminophen, aspirin, ibuprofen or naproxen), (2) variousconcentrations of lipopolysaccharide (LPS), which is an effector ofinflammatory stimuli to macrophage cells, (3) various concentrations ofcarbachol or acetylcholine, which are effectors of non-inflammatorystimuli, (4) analgesic and LPS or (5) analgesic and carbachol oracetylcholine. Briefly, the analgesics were dissolved in FBS-freeculture medium (i.e RPMI 1640 supplemented with 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin) anddiluted to desired concentrations by serial dilution with the samemedium. For cells treated with analgesic in the absence of LPS, 50 μl ofanalgesic solution and 50 μl of FBS-free culture medium were added toeach well. For cells treated with analgesic in the presence of LPS, 50μl of analgesic solution and 50 μl of LPS (from Salmonella typhimurium)in FBS-free culture medium were added to each well. All conditions weretested in duplicates.

After 24 or 48 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris and stored at −70° C. for analysis of cytokine responses byELISA. The cells were collected and washed by centrifugation (5 min at1,500 rpm at 4° C.) in 500 μl A of Phosphate buffer (PBS). Half of thecells were then snapped frozen in liquid nitrogen and stored at −70° C.The remaining cells were stained with fluorescent monoclonal antibodiesand analyzed by flow cytometry.

Flow Cytometry Analysis of Co-stimulatory Molecule Expression

For flow cytometry analysis, macrophages were diluted in 100 μl of FACSbuffer (phosphate buffered saline (PBS) with 2% bovine serum albumin(BSA) and 0.01% NaN₃) and stained 30 min at 4° C. by addition ofFITC-conjugated anti-CD40, PE-conjugated anti-CD80, PE-conjugatedanti-CD86 antibody, anti MHC class II (I-A^(d)) PE (BD Bioscience).Cells were then washed by centrifugation (5 min at 1,500 rpm at 4° C.)in 300 μl of FACS buffer. After a second wash, cells were re-suspendedin 200 μl of FACS buffer and the percentage of cells expressing a givenmarker (single positive), or a combination of markers (double positive)were analyzed with the aid of an Accuri C6 flow cytometer (BDBiosciences).

Analysis of Cytokine Responses by ELISA

Culture supernatants were subjected to cytokine-specific ELISA todetermine IL-1β, IL-6, and TNF-α responses in cultures of macrophagestreated with analgesic, LPS alone or a combination of LPS and analgesic.The assays were performed on Nunc MaxiSorp Immunoplates (Nunc) coatedovernight with 100 μl of anti-mouse IL-6, TNF-α mAbs (BD Biosciences) orIL-1β mAb (R&D Systems) in 0.1 M sodium bicarbonate buffer (pH 9.5).After two washes with PBS (200 μl per well), 200 μl of PBS 3% BSA wereadded in each well (blocking) and the plates incubated for 2 hours atroom temperature. Plates were washed again two times by addition of 200μl per well, 100 μl of cytokine standards and serial dilutions ofculture supernatants were added in duplicate, and the plates wereincubated overnight at 4° C. Finally, the plates were washed twice andincubated with 100 μl of secondary biotinylated anti-mouse IL-6, TNFαmAbs (BD Biosciences), or IL-1β (R&D Systems) followed byperoxidase-labeled goat anti-biotin mAb (Vector Laboratories). Thecolorimetric reaction was developed by the addition of 2,2′-azino-bis(3)-ethylbenzylthiazoline-6-sulfonic acid (ABTS) substrate and H₂O₂(Sigma) and the absorbance measured at 415 nm with a Victor® Vmultilabel plate reader (PerkinElmer).

Determination of COX2 Activity and the Production of cAMP and cGMP

The COX2 activity in the cultured macrophages is determined bysequential competitive ELISA (R&D Systems). The production of cAMP andcGMP is determined by the cAMP assay and cGMP assay. These assays areperformed routinely in the art.

Table 1 summarizes the experiments performed with Raw 264 macrophagecell line and main findings in terms of the effects of analgesics oncell surface expression of costimulatory molecules CD40 and CD80.Expression of these molecules is stimulated by COX2 and inflammatorysignals and, thus, was evaluated to determine functional consequences ofinhibition of COX2.

As shown in Table 2, acetaminophen, aspirin, ibuprofen, and naproxeninhibit basal expression of co-stimulatory molecules CD40 and CD80 bymacrophages at all the tested doses (i.e., 5×10⁵ nM, 5×10⁴ nM, 5×10³ nM,5×10² nM, 50 nM, and 5 nM), except for the highest dose (i.e., 5×10⁶nM), which appears to enhance, rather than inhibit, expression of theco-stimulatory molecules. As shown in FIGS. 1A and 1B, such inhibitoryeffect on CD40 and CD50 expression was observed at analgesic doses aslow as 0.05 nM (i.e., 0.00005 μM). This finding supports the notion thata controlled release of small doses of analgesic may be preferable toacute delivery of large doses. The experiment also revealed thatacetaminophen, aspirin, ibuprofen, and naproxen have a similarinhibitory effect on LPS induced expression of CD40 and CD80.

TABLE 1 Summary of experiments LPS Salmonella Control typhimuriumAcetaminophen Aspirin Ibuprofen Naproxen TESTS 1 X 2 X Dose responses(0, 5, 50, 1000) ng/mL 3 X Dose responses (0, 5, 50, 500, 5 × 10³, 5 ×10⁴, 5 × 10⁵, 5 × 10⁶) nM 4 X X (5 ng/mL) Dose responses X (50 ng/mL (0, 5, 50, 500, 5 × 10³, 5 × 10⁴, 5 × 10⁵, 5 × 10⁶) nM X (1000 ng/mL)ANALYSIS a Characterization of activation/stimulatory status: Flowcytometry analysis of CD40, CD80, CD86, and MHC class II b Mediators ofinflammatory responses: ELISA analysis of IL-1β, IL-6, TNF-α

TABLE 2 Summary of main findings Negative LPS5 Dose analgesic (nM)Effectors % Positive Control ng/ml 5 × 10⁶ 5 × 10⁵ 5 × 10⁴ 5 × 10³ 50050 5 CD40⁺CD80⁺ 20.6 77.8 Acetaminophen CD40⁺CD80⁺ 63 18 12 9.8 8.3 9.57.5 Aspirin CD40⁺CD80⁺ 44 11 10.3 8.3 8 10.5 7.5 Ibuprofen CD40⁺CD80⁺ND* 6.4 7.7 7.9 6.0 4.9 5.8 Naproxen CD40⁺CD80⁺ 37 9.6 7.7 6.9 7.2 6.85.2 Analgesic plus LPS Acetaminophen CD40⁺CD80⁺ 95.1 82.7 72.4 68.8 66.866.2 62.1 Aspirin CD40⁺CD80⁺ 84.5 80 78.7 74.7 75.8 70.1 65.7 IbuprofenCD40⁺CD80⁺ ND  67 77.9 72.9 71.1 63.7 60.3 Naproxen CD40⁺CD80⁺ 66.0 74.177.1 71.0 68.8 72 73 *ND: not done (toxicity)

Table 3 summarizes the results of several studies that measured serumlevels of analgesic after oral therapeutic doses in adult humans. Asshown in Table 3, the maximum serum levels of analgesic after an oraltherapeutic dose are in the range of 10⁴ to 10⁵ nM. Therefore, the dosesof analgesic tested in vitro in Table 2 cover the range ofconcentrations achievable in vivo in humans.

TABLE 3 Serum levels of analgesic in human blood after oral therapeuticdoses Maximum serum levels after oral Molecular therapeutic dosesAnalgesic drug weight mg/L nM References Acetaminophen 151.16 11-18  7.2× 10⁴-1.19 × 10⁵ BMC Clinical Pharmacology. 2010, 10: 10 (Tylenol)Anaesth Intensive Care. 2011, 39: 242 Aspirin 181.66  30-100 1.65 ×10⁵-5.5 × 10⁵  Disposition of Toxic Drugs and Chemicals in(Acetylsalicylic acid) Man, 8th Edition, Biomedical Public, Foster City,CA, 2008, pp. 22-25 J Lab Clin Med. 1984 Jun; 103: 869 Ibuprofen 206.2924-32 1.16 × 10⁵-1.55 × 10⁵ BMC Clinical Pharmacology 2010, 10: 10(Advil, Motrin) J Clin Pharmacol. 2001, 41: 330 Naproxen 230.26 Up to Upto J Clin Pharmacol. 2001, 41: 330 (Aleve) 60 2.6 × 10⁵

EXAMPLE 3 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Mouse Bladder Smooth Muscle Cell Responses toInflammatory and Non-Inflammatory Stimuli

Experimental Design

This study is designed to characterize how the optimal doses ofanalgesics determined in Example 2 affect bladder smooth muscle cells incell culture or tissue cultures, and to address whether differentclasses of analgesics can synergize to more efficiently inhibit COX2 andPGE2 responses.

The effectors, analgesic agents and antimuscarinic agents are describedin Example 2.

Primary culture of mouse bladder smooth muscle cells are subjected toshort term (1-2 hrs) or long term (24-48 hrs) stimulation with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

The cells are then analyzed for the release of PGH₂; PGE; PGE₂;Prostacydin; Thromboxane; IL-1β; IL-6; TNF-α; the COX2 activity; theproduction of cAMP and cGMP; the production of IL-1β, IL-6, TNF-α, andCOX2 mRNA; and surface expression of CD80, CD86, and MEW class IImolecules.

Materials and Methods

Isolation and Purification of Mouse Bladder Cells

Bladder cells were removed from euthanized animals C57BL/6 mice (8-12weeks old), and cells were isolated by enzymatic digestion followed bypurification on a Percoll gradient. Briefly, bladders from 10 mice wereminced with scissors to fine slurry in 10 ml of digestion buffer (RPMI1640, 2% fetal bovine serum, 0.5 mg/ml collagenase, 30 μg/ml DNase).Bladder slurries were enzymatically digested for 30 minutes at 37° C.Undigested fragments were further dispersed through a cell-trainer. Thecell suspension was pelleted and added to a discontinue 20%, 40%. and75% Percoll gradient for purification on mononuclear cells. Eachexperiment used 50-60 bladders.

After washes in RPMI 1640, bladder cells were resuspended RPMI 1640supplemented with 10% fetal bovine serum, 15 mM HEPES, 2 mM L-glutamine,100 U/ml penicillin, and 100 μg/ml of streptomycin and seeded inclear-bottom black 96-well cell culture microculture plates at a celldensity of 3×10⁴ cells per well in 100 μl. Cells were cultured at 37° C.in a 5% CO₂ atmosphere.

In Vitro Treatment of Cells with Analgesics

Bladder cells were treated with analgesic solutions (50 μl/well) eitheralone or together with carbachol (10-Molar, 50 μl/well), as an exampleof non-inflammatory stimuli, or lipopolysaccharide (LPS) of Salmonellatyphimurium (1 μg/ml, 50 μl/well), as an example of non-inflammatorystimuli. When no other effectors were added to the cells, 50 μl of RPMI1640 without fetal bovine serum were added to the wells to adjust thefinal volume to 200 μl.

After 24 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris, and stored at -70° C. for analysis of Prostaglandin E2 (PGE₂)responses by ELISA. Cells were fixed, permeabilized, and blocked fordetection of Cyclooxygenase-2 (COX2) using a fluorogenic substrate. Inselected experiment cells were stimulated 12 hours in vitro for analysisof COX2 responses.

Analysis of COX2 Responses

COX2 responses were analyzed by a Cell-Based ELISA using human/mousetotal COX2 immunoassay (R&D Systems), following the instructions of themanufacturer. Briefly, after cells fixation and permeabilization, amouse anti-total COX2 and a rabbit anti-total GAPDH were added to thewells of the clear-bottom black 96-well cell culture microcultureplates. After incubation and washes, an HRP-conjugated anti-mouse IgGand an AP-conjugated anti-rabbit IgG were added to the wells. Followinganother incubation and set of washes, the HRP- and AP-fluorogenicsubstrates were added. Finally, a Victor® V multilabel plate reader(PerkinElmer) was used to read the fluorescence emitted at 600 nm (COX2fluorescence) and 450 nm (GAPDH fluorescence). Results are expressed asrelative levels of total COX2 as determined by relative fluorescenceunit (RFUs) and normalized to the housekeeping protein GAPDH.

Analysis of PGE2 Responses

Prostaglandin E2 responses were analyzed by a sequential competitiveELISA (R&D Systems). More specifically, culture supernatants or PGE2standards were added to the wells of a 96-well polystyrene microplatecoated with a goat anti-mouse polyclonal antibody. After one hourincubation on a microplate shaker, an HRP-conjugated PGE2 was added andthe plates were incubated for an additional two hours at roomtemperature. The plates were then washed and HRP substrate solutionadded to each well. The color was allowed to develop for 30 minutes, andthe reaction stopped by the addition of sulfuric acid before reading theplate at 450 nm with wavelength correction at 570 nm. Results areexpressed as mean pg/ml of PGE2.

Other Assays

The release of PGH₂; PGE, Prostacydin; Thromboxane; IL-1β; IL-6; andTNF-α; the production of cAMP and cGMP; the production of IL-1β, IL-6,TNF-α, and COX2 mRNA; and surface expression of CD80, CD86, and MHCclass II molecules are determined as described in Example 2.

Results

Analgesics Inhibit COX2 Responses of Mouse Bladder Cells to anInflammatory Stimulus

Several analgesics (acetaminophen, aspirin, ibuprofen, and naproxen)were tested on mouse bladder cells at the concentration of 5 μM or 50 μMto determine whether the analgesics could induce COX2 responses.Analysis of 24-hour cultures showed that none of the analgesics testedinduced COX2 responses in mouse bladder cells in vitro.

The effect of these analgesics on the COX2 responses of mouse bladdercells to carbachol or LPS stimulation in vitro was also tested. Asindicated in Table 1, the dose of carbachol tested has no significanteffect on COX2 levels in mouse bladder cells. On the other hand, LPSsignificantly increased total COX2 levels. Interestingly, acetaminophen,aspirin, ibuprofen, and naproxen could all suppress the effect of LPS onCOX2 levels. The suppressive effect of the analgesic was seen when thesedrugs were tested at either 5 μM or 50 μM (Table 4).

TABLE 4 COX2 expression by mouse bladder cells after in vitrostimulation and treatment with analgesic Total COX2 levels StimulusAnalgesic (Normalized RFUs) None None 158 ± 18 Carbachol (mM) None 149 ±21 LPS (1 μg/ml) None 420 ± 26 LPS (1 μg/ml) Acetaminophen (5 μM) 275 ±12 LPS (1 μg/ml) Aspirin (5 μM) 240 ± 17 LPS (1 μg/ml) Ibuprofen (5 μM))253 ± 32 LPS (1 μg/ml) Naproxen (5 μM) 284 ± 11 LPS (1 μg/ml)Acetaminophen (50 μM) 243 ± 15 LPS (1 μg/ml) Aspirin (50 μM) 258 ± 21LPS (1 μg/ml) Ibuprofen (50 μM) 266 ± 19 LPS (1 μg/ml) Naproxen (50 μM)279 ± 23Analgesics Inhibit PGE2 Responses of Mouse Bladder Cells to anInflammatory Stimulus

The secretion of PGE2 in culture supernatants of mouse bladder cells wasmeasured to determine the biological significance of the alteration ofmouse bladder cell COX2 levels by analgesics. As shown in Table 5, PGE2was not detected in the culture supernatants of unstimulated bladdercells or bladder cells cultured in the presence of carbachol. Consistentwith COX2 responses described above, stimulation of mouse bladder cellswith LPS induced the secretion of high levels of PGE2. Addition of theanalgesics acetaminophen, aspirin, ibuprofen, and naproxen suppressedthe effect of LPS on PGE2 secretion, and no difference was seen betweenthe responses of cells treated with the 5 or 50 μM dose of analgesic.

TABLE 5 PGE2 secretion by mouse bladder cells after in vitro stimulationand treatment with analgesic. Stimulus Analgesic PGE2 levels (pg/ml)None None <20.5 Carbachol (mM) None <20.5 LPS (1 μg/ml) None 925 ± 55LPS (1 μg/ml) Acetaminophen (5 μM) 619 ± 32 LPS (1 μg/ml) Aspirin (5 μM)588 ± 21 LPS (1 μg/ml) Ibuprofen (5 μM)) 593 ± 46 LPS (1 μg/ml) Naproxen(5 μM) 597 ± 19 LPS (1 μg/ml) Acetaminophen (50 μM) 600 ± 45 LPS (1μg/ml) Aspirin (50 μM) 571 ± 53 LPS (1 μg/ml) Ibuprofen (50 μM) 568 ± 32LPS (1 μg/ml) Naproxen (50 μM) 588 ± 37

In summary, these data show that the analgesics alone at 5 μM or 50 μMdo not induce COX2 and PGE2 responses in mouse bladder cells. Theanalgesics at 5 μM or 50 μM, however, significantly inhibit COX2 andPGE2 responses of mouse bladder cells stimulated in vitro with LPS (1μg/ml). No significant effect of analgesics was observed on COX2 andPGE2 responses of mouse bladder cells stimulated with carbachol (1 mM).

EXAMPLE 4 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Mouse Bladder Smooth Muscle Cell Contraction

Experimental Design

Cultured mouse or rat bladder smooth muscle cells and mouse or ratbladder smooth muscle tissue are exposed to inflammatory stimuli andnon-inflammatory stimuli in the presence of analgesic agent and/orantimuscarinic agent at various concentrations. The stimulus-inducedmuscle contraction is measured to evaluate the inhibitory effect of theanalgesic agent and/or antimuscarinic agent.

The effectors, analgesic agents, and antimuscarinic agents are describedin Example 2.

Primary cultures of mouse bladder smooth muscle cells are subjected toshort term (1-2 hrs) or long term (24-48 hrs) stimulation with:

-   -   (1) Each analgesic agent alone at various doses.    -   (2) Each analgesic agent at various doses in the presence of        LPS.    -   (3) Each analgesic agent at various doses in the presence of        carbachol or acetylcholine.    -   (4) Each analgesic agent at various doses in the presence of AA,        DGLA, or EPA.    -   (5) Botulinum neurotoxin A alone at various doses.    -   (6) Botulinum neurotoxin A at various doses in the presence of        LPS.    -   (7) Botulinum neurotoxin A at various doses in the presence of        carbachol or acetylcholine.    -   (8) Botulinum neurotoxin A at various doses in the presence of        AA, DGLA, or EPA.    -   (9) Each antimuscarinic agent alone at various doses.    -   (10) Each antimuscarinic agent at various doses in the presence        of LPS.    -   (11) Each antimuscarinic agent at various doses in the presence        of carbachol or acetylcholine.    -   (12) Each antimuscarinic agent at various doses in the presence        of AA, DGLA, or EPA.        Materials and Methods

Primary mouse bladder cells are isolated as described in Example 3. Inselected experiments, cultures of bladder tissue are used. Bladdersmooth muscle cell contractions are recorded with a Grass polygraph(Quincy Mass, USA).

EXAMPLE 5 Effect of Oral Analgesic Agents and Antimuscarinic Agents onCox2 and Pge2 Responses of Mouse Bladder Smooth Muscle Cells

Experimental Design

Normal mice and mice with over active bladder syndrome are given oraldoses of aspirin, naproxen sodium, ibuprofen, Indocin, nabumetone,Tylenol, Celecoxib, oxybutynin, solifenacin, darifenacin, atropine, andcombinations thereof. Control groups include untreated normal mice anduntreated OAB mice with over active bladder syndrome. Thirty (30)minutes after last doses, the bladders are collected and stimulated exvivo with carbachol or acetylcholine. In selected experiments, thebladders are treated with botulinum neurotoxin A before stimulation withcarbachol. Animals are maintained in metabolic cages and frequency (andvolume) of urination are evaluated. Bladder outputs are determined bymonitoring water intake and cage litter weight. Serum PGH₂, PGE, PGE₂,Prostacydin, Thromboxane, IL-1β, IL-6, TNF-α, cAMP, and cGMP levels aredetermined by ELISA. CD80, CD86, and MHC class II expression in wholeblood cells are determined by flow cytometry.

At the end of the experiment, animals are euthanized, and ex vivobladder contractions are recorded with a Grass polygraph. Portions ofbladders are fixed in formalin, and COX2 responses are analyzed byimmunohistochemistry.

EXAMPLE 6 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Human Bladder Smooth Muscle Cell Responses toInflammatory and Non-Inflammatory Stimuli

Experimental Design

This study is designed to characterize how the optimal doses ofanalgesic determined in Examples 1-5 affect human bladder smooth musclecells in cell culture or tissue cultures and to address whetherdifferent classes of analgesics can synergize to more efficientlyinhibit COX2 and PGE2 responses.

The effectors, analgesic agents, and antimuscarinic agents are describedin Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs)or long term (24-48 hrs) stimulation with:

-   -   (1) Each analgesic agent alone at various doses.    -   (2) Each analgesic agent at various doses in the presence of        LPS.    -   (3) Each analgesic agent at various doses in the presence of        carbachol or acetylcholine.    -   (4) Each analgesic agent at various doses in the presence of AA,        DGLA, or EPA.    -   (5) Botulinum neurotoxin A alone at various doses.    -   (6) Botulinum neurotoxin A at various doses in the presence of        LPS.    -   (7) Botulinum neurotoxin A at various doses in the presence of        carbachol or acetylcholine.    -   (8) Botulinum neurotoxin A at various doses in the presence of        AA, DGLA, or EPA.    -   (9) Each antimuscarinic agent alone at various doses.    -   (10) Each antimuscarinic agent at various doses in the presence        of LPS.    -   (11) Each antimuscarinic agent at various doses in the presence        of carbachol or acetylcholine.    -   (12) Each antimuscarinic agent at various doses in the presence        of AA, DGLA, or EPA.

The cells are then analyzed for the release of PGH₂; PGE; PGE₂;Prostacydin; Thromboxane; IL-1β; IL-6; TNFa; the COX2 activity; theproduction of cAMP and cGMP; the production of IL-1β, IL-6, TNFa, andCOX2 mRNA; and surface expression of CD80, CD86, and MHC class IImolecules.

EXAMPLE 7 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Human Bladder Smooth Muscle Cell Contraction

Experimental Design

Cultured human bladder smooth muscle cells are exposed to inflammatorystimuli and non-inflammatory stimuli in the presence of an analgesicagent and/or antimuscarinic agent at various concentrations. Thestimuli-induced muscle contraction is measured to evaluate theinhibitory effect of the analgesic agent and/or antimuscarinic agent.

The effectors, analgesic agents, and antimuscarinic agents are describedin Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs)or long term (24-48 hrs) stimulation with:

-   -   (1) Each analgesic agent alone at various doses.    -   (2) Each analgesic agent at various doses in the presence of        LPS.    -   (3) Each analgesic agent at various doses in the presence of        carbachol or acetylcholine.    -   (4) Each analgesic agent at various doses in the presence of AA,        DGLA, or EPA.    -   (5) Botulinum neurotoxin A alone at various doses.    -   (6) Botulinum neurotoxin A at various doses in the presence of        LPS.    -   (7) Botulinum neurotoxin A at various doses in the presence of        carbachol or acetylcholine.    -   (8) Botulinum neurotoxin A at various doses in the presence of        AA, DGLA, or EPA.    -   (9) Each antimuscarinic agent alone at various doses.    -   (10) Each antimuscarinic agent at various doses in the presence        of LPS.    -   (11) Each antimuscarinic agent at various doses in the presence        of carbachol or acetylcholine.    -   (12) Each antimuscarinic agent at various doses in the presence        of AA, DGLA, or EPA.

Bladder smooth muscle cell contractions are recorded with a Grasspolygraph (Quincy Mass, USA).

EXAMPLE 8 Effect of Analgesic Agents on Normal Human Bladder SmoothMuscle Cell Responses to Inflammatory and Non Inflammatory Signals

Experimental Design

Culture of Normal Human Bladder Smooth Muscle Cells

Normal human bladder smooth muscle cells were isolated by enzymaticdigestion from macroscopically normal pieces of human bladder. Cellswere expended in vitro by culture at 37° C. in a 5% CO₂ atmosphere inRPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 mg/ml of streptomycin andpassage once a week by treatment with trypsin to detach cells followedby reseeding in a new culture flask. The first week of culture, theculture medium was supplemented with 0.5 ng/ml epidermal growth factor,2 ng/ml fibroblast growth factor, and 5 μg/ml insulin.

Treatment of Normal Human Bladder Smooth Muscle Cells with Analgesics InVitro

Bladder smooth muscle cells trypsinized and seeded in microcultureplates at a cell density of 3×10⁴ cells per well in 100 μl were treatedwith analgesic solutions (50 μl/well) either alone or together carbachol(10-Molar, 50 μl/well), as an example of non-inflammatory stimuli, orlipopolysaccharide (LPS) of Salmonella typhimurium (1 μg/ml, 50μl/well), as an example of non-inflammatory stimuli. When no othereffectors were added to the cells, 50 μl of RPMI 1640 without fetalbovine serum were added to the wells to adjust the final volume to 200μl.

After 24 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris, and stored at −70° C. for analysis of Prostaglandin E2 (PGE₂)responses by ELISA. Cells were fixed, permeabilized, and blocked fordetection of COX2 using a fluorogenic substrate. In selectedexperiments, cells were stimulated 12 hours in vitro for analysis ofCOX2, PGE2, and cytokine responses.

Analysis of COX2, PGE2, and Cytokine Responses

COX2 and PGE2 responses were analyzed as described in Example 3.Cytokine responses were analyzed as described in Example 2.

Results

Analgesics inhibit COX2 responses of normal human bladder smooth musclecells to inflammatory and non-inflammatory stimuli—Analysis of cells andculture supernatants after 24 hours of cultures showed that none of theanalgesics tested alone induced COX2 responses in normal human bladdersmooth muscle cells. However, as summarized in Table 6, carbacholinduced low, but significant COX2 responses in normal human bladdersmooth muscle cells. On the other hand, LPS treatment resulted in higherlevels of COX2 responses in normal human bladder smooth muscle cells.Acetaminophen, aspirin, ibuprofen, and naproxen could all suppress theeffect of carbachol and LPS on COX2 levels. The suppressive effect ofthe analgesics was seen on LPS-induced responses when these drugs weretested at either 5 μM or 50 μM.

TABLE 6 COX2 expression by normal human bladder smooth muscle cellsafter in vitro stimulation with inflammatory and non- inflammatorystimuli and treatment with analgesic Total COX2 Total levels^(#) COX2levels (Normalized (Normalized RFUs) RFUs) Stimulus Analgesic subject 1subject 2 None None 230 199 Carbachol 10⁻³M None (50 μM) 437 462Carbachol 10⁻³M Acetaminophen (50 μM) 298 310 Carbachol 10⁻³M Aspirin(50 μM) 312 297 Carbachol 10⁻³M Ibuprofen (50 μM) 309 330 Carbachol10⁻³M Naproxen (50 μM) 296 354 LPS (10 μg/ml) None 672 633 LPS (10μg/ml) Acetaminophen (5 μM) 428 457 LPS (10 μg/ml) Aspirin (5 μM) 472491 LPS (10 μg/ml) Ibuprofen (5 μM) 417 456 LPS (10 μg/ml) Naproxen (5μM 458 501 LPS (10 μg/ml) Acetaminophen (50 μM) 399 509 LPS (10 μg/ml)Aspirin (50 μM) 413 484 LPS (10 μg/ml) Ibuprofen (50 μM) 427 466 LPS (10μg/ml) Naproxen (50 μM) 409 458 ^(#)Data are expressed as mean ofduplicates

Analgesics inhibit PGE2 responses of normal human bladder smooth musclecells to inflammatory and non-inflammatory stimuli—Consistent with theinduction of COX2 responses described above, both carbachol and LPSinduced production of PGE2 by normal human bladder smooth muscle cells.Acetaminophen, aspirin, ibuprofen, and naproxen were also found tosuppress the LPS-induced PGE2 responses at either 5 μM or 50 μM (Table7).

TABLE 7 PGE2 secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non- inflammatory stimuli andtreatment with analgesic PGE2 levels^(#) PGE2 levels (pg/ml) (pg/ml)Stimulus Analgesic Subject 1 Subject 2 None None <20.5 <20.5 Carbachol10⁻³M None 129 104 Carbachol 10⁻³M Acetaminophen (50 μM) 76 62 Carbachol10⁻³M Aspirin (50 μM) 89 59 Carbachol 10⁻³M Ibuprofen (50 μM) 84 73Carbachol 10⁻³M Naproxen (50 μM) 77 66 LPS (10 μg/ml) None 1125 998 LPS(10 μg/ml) Acetaminophen (5 μM) 817 542 LPS (10 μg/ml) Aspirin (5 μM)838 598 LPS (10 μg/ml) Ibuprofen (5 μM) 824 527 LPS (10 μg/ml) Naproxen(5 μM 859 506 LPS (10 μg/ml) Acetaminophen (50 μM) 803 540 LPS (10μg/ml) Aspirin (50 μM) 812 534 LPS (10 μg/ml) Ibuprofen (50 μM) 821 501LPS (10 μg/ml) Naproxen (50 μM) 819 523 ^(#)Data are expressed as meanof duplicates

Analgesics inhibit cytokine responses of normal human bladder cells toinflammatory stimuli—Analysis of cells and culture supernatants after 24hours of culture showed that none of the analgesics tested alone inducedIL-6 or TNFα secretion in normal human bladder smooth muscle cells. Asshown in Tables 8 and 9, the doses of carbachol tested induced low, butsignificant TNFa and IL-6 responses in normal human bladder smoothmuscle cells. On the other hand, LPS treatment resulted in massiveinduction of these proinflammatory cytokines. Acetaminophen, aspirin,ibuprofen, and naproxen suppress the effect of carbachol and LPS on TNFαand IL-6 responses. The suppressive effect of the analgesics onLPS-induced responses was seen when these drugs were tested at either 5μM or 50 μM.

TABLE 8 TNFα secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic TNFα TNFα (pg/ml)^(#) (pg/ml) Stimuli AnalgesicSubject 1 Subject 2 None None <5 <5 Carbachol 10⁻³M None 350 286Carbachol 10⁻³M Acetaminophen (50 μM) 138 164 Carbachol 10⁻³M Aspirin(50 μM) 110 142 Carbachol 10⁻³M Ibuprofen (50 μM) 146 121 Carbachol10⁻³M Naproxen (50 μM) 129 137 LPS (10 μg/ml) None 5725 4107 LPS (10μg/ml) Acetaminophen (5 μM) 2338 2267 LPS (10 μg/ml) Aspirin (5 μM) 24792187 LPS (10 μg/ml) Ibuprofen (5 μM) 2733 2288 LPS (10 μg/ml) Naproxen(5 μM 2591 2215 LPS (10 μg/ml) Acetaminophen (50 μM) 2184 2056 LPS (10μg/ml) Aspirin (50 μM) 2266 2089 LPS (10 μg/ml) Ibuprofen (50 μM) 26031997 LPS (10 μg/ml) Naproxen (50 μM) 2427 2192 ^(#)Data are expressed asmean of duplicates.

TABLE 9 IL-6 secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic IL-6 IL-6 (pg/ml)^(#) (pg/ml) StimulusAnalgesic Subject 1 Subject 2 None None <5 <5 Carbachol 10⁻³M None 232278 Carbachol 10⁻³M Acetaminophen (50 μM) 119 135 Carbachol 10⁻³MAspirin (50 μM) 95 146 Carbachol 10⁻³M Ibuprofen (50 μM) 107 118Carbachol 10⁻³M Naproxen (50 μM) 114 127 LPS (10 μg/ml) None 4838 4383LPS (10 μg/ml) Acetaminophen (5 μM) 2012 2308 LPS (10 μg/ml) Aspirin (5μM) 2199 2089 LPS (10 μg/ml) Ibuprofen (5 μM) 2063 2173 LPS (10 μg/ml)Naproxen (5 μM 2077 2229 LPS (10 μg/ml) Acetaminophen (50 μM) 2018 1983LPS (10 μg/ml) Aspirin (50 μM) 1987 2010 LPS (10 μg/ml) Ibuprofen (50μM) 2021 1991 LPS (10 μg/ml) Naproxen (50 μM) 2102 2028 ^(#)Data areexpressed as mean of duplicates

Primary normal human bladder smooth muscle cells were isolated, culturedand evaluated for their responses to analgesics in the presence ofnon-inflammatory (carbachol) and inflammatory (LPS) stimuli. The goal ofthis study was to determine whether or not normal human bladder smoothmuscle cells recapitulate the observations previously made with murinebladder cells.

The above-described experiment will be repeated with analgesic agentsand/or antimuscarinic agents in delayed-release, or extended-releaseformulation or delayed-and-extended-release formulations.

The above description is for the purpose of teaching a person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. A phatinaceutical composition, comprising: afirst component comprising one or more therapeutically activeingredients formulated for immediate-release after oral administration;and a second component comprising a first subcomponent comprising one ormore therapeutically active ingredients formulated for immediate-releaseafter gastric emptying-and a second subcomponent comprising one or moretherapeutically active ingredient formulated for extended-release aftergastric emptying, wherein each therapeutically active ingredient in thefirst and second components is an analgesic agent or a prostaglandinpathway inhibitor, wherein analgesic agents and prostaglandin pathwayinhibitors are the only therapeutically active ingredients in thepharmaceutical composition, and wherein none of the prostaglandinpathway inhibitor(s) includes acetaminophen or a non-steroidalanti-inflammatory drug (NSAID).
 2. The pharmaceutical composition ofclaim 1, wherein the analgesic agents are selected from the groupconsisting of aspirin, ibuprofen, naproxen, naproxen sodium,indomethacin, nabumetone, and acetaminophen.
 3. The pharmaceuticalcomposition of claim 1, wherein the second component is formulated torelease the subcomponents after a lag time of 1-4 hours following oraladministration.
 4. The pharmaceutical composition of claim 1, whereinthe first component further comprises an extended-release subcomponent,wherein the extended-release subcomponent comprises one or moreanalgesic agents and/or prostaglandin pathway inhibitors.
 5. Thepharmaceutical composition of claim 1, wherein the first componentcomprises acetaminophen and a prostaglandin pathway inhibitor.
 6. Thepharmaceutical composition of claim 5, wherein each of the first andsecond subcomponents of the second component comprises acetaminophen anda prostaglandin pathway inhibitor.
 7. The pharmaceutical composition ofclaim 1, wherein the first component comprises ibuprofen and aprostaglandin pathway inhibitor.
 8. The pharmaceutical composition ofclaim 7, wherein each of the first and second subcomponents of thesecond component comprises ibuprofen and a prostaglandin pathwayinhibitor.
 9. The pharmaceutical composition of claim 1, wherein thefirst component comprises ibuprofen, acetaminophen and a prostaglandinpathway inhibitor.
 10. The pharmaceutical composition of claim 9,wherein each of the first and second subcomponents of the secondcomponent comprises ibuprofen, acetaminophen and a prostaglandin pathwayinhibitor.